Sequential Isolation of Microglia and Astrocytes from Young and Aged Adult Mouse Brains for Downstream Transcriptomic Analysis

In aging, the brain is more vulnerable to injury and neurodegenerative disease, but the mechanisms responsible are largely unknown. Evidence now suggests that neuroinflammation, mediated by resident brain astrocyte and microglia populations, are key players in the generation of inflammatory responses and may influence both age related processes and the initiation/progression of neurodegeneration. Consequently, targeting these cell types individually and collectively may aid in the development of novel disease-modifying therapies. We have optimized and characterized a protocol for the effective sequential isolation of both microglia and astrocytes from the adult mouse brain in young and aged mice. We demonstrate a technique for the sequential isolation of these immune cells by using magnetic beads technology, optimized to increase yield and limit potential artifacts in downstream transcriptomic applications, including RNA-sequencing pipelines. This technique is versatile, cost-effective, and reliable for the study of responses within the same biological context, simultaneously being advantageous in reducing mice numbers required to assess cellular responses in normal and age-related pathological conditions.


Introduction
Microglia and astrocytes are key cellular components of the central nervous system (CNS), contributing to a variety of processes such as homeostasis, blood-brain barrier maintenance, synaptic remodeling and functional support of neurons [1,2]. Not only are these cells vital for proper CNS function, they are also resident immune cells that can initiate and sustain neuroinflammation, a key factor in the pathogenesis of many agerelated neurodegenerative disorders. Activation of microglia and astrocytes in various injury or age-related paradigms can result in the secretion of cytokines, chemokines, and recruitment of circulating immune cells [3,4]. Temporally, these immune responses largely overlap and are crucial for repair, however if uncontrolled, these inflammatory responses exacerbate CNS injury and neurodegenerative pathology [5,6]. Despite knowledge of both the reparative and deleterious roles of these cells, heterogeneity occurs depending on disease paradigm, location, and spatial interaction between each other. Therefore, sequential isolation of microglia and astrocytes using fast isolation techniques that provide pure and sufficient cell specific yields without interference from other cell contaminants, is a powerful tool to study cellular immunophenotypes within the same brain tissue in models of neurodegeneration.
To achieve this in adult mouse brains, fluorescent-activated cell sorting (FACs) is a commonly used approach. FACs uses fluorescently labelled antibodies to identify subpopulations of cells in large numbers and at high purity [7,8]. However, it has its own inherent disadvantages. It requires a large starting number of cells in suspension, and as a result it may fail to isolate sufficient single cells from low quantity cell populations or small tissue samples [9]. The rapid flow of solution through the machine and non-specific fluorescent

Euthanasia and Transcardiac Perfusion
Note: Follow approved procedures of anesthesia and euthanasia in accordance with protocols approved with your relevant institution. The following is performed in accordance with protocols approved by the Georgetown University Animal Care and Use Committee.
1. Euthanize mouse (C57Bl/6J) via CO2 chamber at a flow rate of 4 L/min for approximately 3 min; 2. Place mouse on ice and to lift the upper ventral abdomen using Graefe forceps; 3. While holding Graefe forceps, make cutaneal incisions with light operating scissors, 3 cm horizontally and 3 cm vertically from the abdominal midline to expose the peritoneal cavity; 4. After cutaneal incisions have been made, confirm secondary euthanasia by lifting the xyphoid process with Graefe forceps and creating lateral incisions of the diaphragm along the upper thoracic line; 5. Make bilateral incisions of the thoracic vertebra and using Halsted mosquito forceps hold the xyphoid process above the chest cavity; 6. With the heart now exposed, insert a 21-gauge needle 5 mm into the left ventricle; 7. Turn on peristaltic pump to initiate transcardiac flush of ice-cold 1X PBS at 5 mL/min (see Table 3); 8. After turning on the pump, immediately incise the right atrium by 3 mm using micro dissecting scissors (see Figure 2); 9. Continue perfusion for approximately 2 min or until visible color change of liver indicating successful perfusion.

Euthanasia and Transcardiac Perfusion
Note: Follow approved procedures of anesthesia and euthanasia in accordance with protocols approved with your relevant institution. The following is performed in accordance with protocols approved by the Georgetown University Animal Care and Use Committee.
Place mouse on ice and to lift the upper ventral abdomen using Graefe forceps; 3.
While holding Graefe forceps, make cutaneal incisions with light operating scissors, 3 cm horizontally and 3 cm vertically from the abdominal midline to expose the peritoneal cavity; 4.
After cutaneal incisions have been made, confirm secondary euthanasia by lifting the xyphoid process with Graefe forceps and creating lateral incisions of the diaphragm along the upper thoracic line; 5.
Make bilateral incisions of the thoracic vertebra and using Halsted mosquito forceps hold the xyphoid process above the chest cavity; 6.
With the heart now exposed, insert a 21-gauge needle 5 mm into the left ventricle; 7.
Turn on peristaltic pump to initiate transcardiac flush of ice-cold 1X PBS at 5 mL/min (see Table 3); 8.
After turning on the pump, immediately incise the right atrium by 3 mm using micro dissecting scissors (see Figure 2); 9.
Continue perfusion for approximately 2 min or until visible color change of liver indicating successful perfusion.
Methods Protoc. 2022, 5, x FOR PEER REVIEW 5 of 16 Figure 2. Schematic workflow of transcardiac perfusion, brain removal, dissection, and preparation of tissue for dissociation and enzymatic digestion. (Red denotes key steps that should be proceeded with carefully to ensure optimal performance, especially if dealing with small tissue quantity <50 mg).

Brain Removal
1. Immediately post perfusion, decapitate the mouse with 6.5" operating scissors; 2. Cut the skin above the midline with micro dissecting scissors, to expose the skull and externally rotate the skin above the head; 3. Make bilateral incisions between the posterior midline and the parietal bone (approximately 10 mm) and between the posterior midline and the occipital bone (approximately 5 mm), creating a t-formation; Figure 2. Schematic workflow of transcardiac perfusion, brain removal, dissection, and preparation of tissue for dissociation and enzymatic digestion. (Red denotes key steps that should be proceeded with carefully to ensure optimal performance, especially if dealing with small tissue quantity <50 mg).
Cut the skin above the midline with micro dissecting scissors, to expose the skull and externally rotate the skin above the head; 3.
Make bilateral incisions between the posterior midline and the parietal bone (approximately 10 mm) and between the posterior midline and the occipital bone (approximately 5 mm), creating a t-formation; 4.
Using closed micro dissecting scissors, insert tip into the junction of the interfrontal suture and frontonasal sutures approximately 2 mm into the skull, then gently open the scissors to crack the calvaria midline down the interfrontal and sagittal suture, opening the skull into two hemispheres; 5.
Remove connective tissue from skull, and remove brain using curved forceps or micro spatula; 7.
Place brain on a cold plate for immediate dissection of brain regions.

1.
Position the brain on ice cold plate so that the cerebral cortices are facing upwards. Using a sterile razor blade, hemisect the brain;

2.
Using one brain hemisphere, turn over with cortex facing the cold block. Using small curved forceps, pinch out striatal/thalamus region leaving only cortical and hippocampal tissue; 3.
Using a sterile scalpel, slice/mince selected micro punch into small pieces; 5.
Place segments of minced micro punch tissue around the cap of gentleMACS C Tube in a circular arrangement; 6.
Place cap with circular arranged tissue on gentleMACS C tube and close; 9.
Tip C tube upside down, ensuring enzyme mix 1 and 2 solution is covering tissue; 10. Place closed C tube with tissue and enzymes on gentleMACS Octo Dissociator to proceed with gentleMACS Program 37C_ABDK_02; Methods Protoc. 2022, 5, x FOR PEER REVIEW 7 of 16 Magnetic Separation with MS Columns for Microglia 8. Place MS columns in the magnetic field of MACS Separator and prepare column by rinsing through 500 µL PB buffer. Discard flow through; CRITICAL STEP Wait until column is completely empty before proceeding to next step; OPTIONAL STEP To remove air bubbles from column after 500 µL rinse, use a needle syringe to pop bubbles; 9. Prepare 5 mL flow through collection tubes underneath MS columns for negative flow through collection; 10. Apply the 500 µL of CD11b + labeled cells (from step 7 above) into MS column attached to magnetic separator; CRITICAL STEP To increase purity of microglia, apply negative flow through from each collection tube back into corresponding MS column for a second time; 11. Wash MS column with 500 µL PB buffer three times; 12. After wash is complete, remove 5 mL collection tubes (containing negative fraction); 13. Place 5 mL collection tubes (containing negative fraction) into a 15 mL falcon to allow for centrifugation; CRITICAL STEP 14. Centrifuge 15 mL falcon containing 5 mL collection tubes with negative flow though at 300× g for 10 min at 4 °C. Set aside for astrocyte labelling in Section 3.6; 15. Remove MS column containing CD11b + bound cells from the separator, place it on corresponding collection tube, add 1 mL PB buffer, and immediately flush out to elute CD11b + magnetically labeled cells with provided plunger; 16. Repeat step 14 with an additional 1 mL of PB buffer to increase yield;

CRITICAL STEP
17. Proceed to RNA extraction of eluted Cd11b + microglia.
CRITICAL STEP Samples should be prepared directly after perfusion for optimal cell suspension performance. Keep samples on ice, especially when preparing larger sample sizes, to maintain optimal tissue integrity.
After termination of the ABDK_02 program, detach C Tube from the gentleMACS Octo Dissociator with Heaters; 3.
Resuspend sample by adding 10 mL of cold D-PBS to the gentleMACS C Tube. Close the tube and gently shake to collect samples at the bottom. Gentle inversions may be necessary to retrieve any remaining tissue on the lid; 4.
Add components of C Tube through MACS SmartStrainers (70 µm) into a 50 mL conical tube; Methods Protoc. 2022, 5, x FOR PEER REVIEW 7 of 16 Magnetic Separation with MS Columns for Microglia 8. Place MS columns in the magnetic field of MACS Separator and prepare column by rinsing through 500 µL PB buffer. Discard flow through; CRITICAL STEP Wait until column is completely empty before proceeding to next step; OPTIONAL STEP To remove air bubbles from column after 500 µL rinse, use a needle syringe to pop bubbles; 9. Prepare 5 mL flow through collection tubes underneath MS columns for negative flow through collection; 10. Apply the 500 µL of CD11b + labeled cells (from step 7 above) into MS column attached to magnetic separator; CRITICAL STEP To increase purity of microglia, apply negative flow through from each collection tube back into corresponding MS column for a second time; 11. Wash MS column with 500 µL PB buffer three times; 12. After wash is complete, remove 5 mL collection tubes (containing negative fraction); 13. Place 5 mL collection tubes (containing negative fraction) into a 15 mL falcon to allow for centrifugation; CRITICAL STEP 14. Centrifuge 15 mL falcon containing 5 mL collection tubes with negative flow though at 300× g for 10 min at 4 °C. Set aside for astrocyte labelling in Section 3.6; 15. Remove MS column containing CD11b + bound cells from the separator, place it on corresponding collection tube, add 1 mL PB buffer, and immediately flush out to elute CD11b + magnetically labeled cells with provided plunger; 16. Repeat step 14 with an additional 1 mL of PB buffer to increase yield; Magnetic Separation with MS Columns for Microglia 8. Place MS columns in the magnetic field of MACS Separator and prepare column by rinsing through 500 µL PB buffer. Discard flow through; CRITICAL STEP Wait until column is completely empty before proceeding to next step; OPTIONAL STEP To remove air bubbles from column after 500 µL rinse, use a needle syringe to pop bubbles; 9. Prepare 5 mL flow through collection tubes underneath MS columns for negative flow through collection; 10. Apply the 500 µL of CD11b + labeled cells (from step 7 above) into MS column attached to magnetic separator; CRITICAL STEP To increase purity of microglia, apply negative flow through from each collection tube back into corresponding MS column for a second time; 11. Wash MS column with 500 µL PB buffer three times; 12. After wash is complete, remove 5 mL collection tubes (containing negative fraction); 13. Place 5 mL collection tubes (containing negative fraction) into a 15 mL falcon to allow for centrifugation; CRITICAL STEP 14. Centrifuge 15 mL falcon containing 5 mL collection tubes with negative flow though at 300× g for 10 min at 4 °C. Set aside for astrocyte labelling in Section 3.6; 15. Remove MS column containing CD11b + bound cells from the separator, place it on corresponding collection tube, add 1 mL PB buffer, and immediately flush out to elute CD11b + magnetically labeled cells with provided plunger; CRITICAL STEP Transfer samples from 50 mL conical tubes to new 15 mL conical tubes after filtration;
Resuspend cell pellet with 1550 µL of cold D-PBS in the 15 mL conical tube; 7.
Add 450 µL of debris removal solution and mix well; 8.
Gently overlay 2 mL of D-PBS into tube. Be careful not to mix phases; OPTIONAL STEP Slowly pipette D-PBS with the conical tube at a 45 • angle to prevent phase mixing;

9.
Centrifuge samples at 300× g for 10 min at 4 • C; 10. After three phases are formed, aspirate the top two phases gently and completely discard; Magnetic Separation with MS Columns for Microglia 8. Place MS columns in the magnetic field of MACS Separator and prepare column by rinsing through 500 µL PB buffer. Discard flow through; CRITICAL STEP Wait until column is completely empty before proceeding to next step; OPTIONAL STEP To remove air bubbles from column after 500 µL rinse, use a needle syringe to pop bubbles; 9. Prepare 5 mL flow through collection tubes underneath MS columns for negative flow through collection; 10. Apply the 500 µL of CD11b + labeled cells (from step 7 above) into MS column attached to magnetic separator; CRITICAL STEP To increase purity of microglia, apply negative flow through from each collection tube back into corresponding MS column for a second time; 11. Wash MS column with 500 µL PB buffer three times; 12. After wash is complete, remove 5 mL collection tubes (containing negative fraction); 13. Place 5 mL collection tubes (containing negative fraction) into a 15 mL falcon to allow for centrifugation; CRITICAL STEP 14. Centrifuge 15 mL falcon containing 5 mL collection tubes with negative flow though at 300× g for 10 min at 4 °C. Set aside for astrocyte labelling in Section 3.6; 15. Remove MS column containing CD11b + bound cells from the separator, place it on corresponding collection tube, add 1 mL PB buffer, and immediately flush out to elute CD11b + magnetically labeled cells with provided plunger; 16. Repeat step 14 with an additional 1 mL of PB buffer to increase yield;

CRITICAL STEP
17. Proceed to RNA extraction of eluted Cd11b + microglia.
CRITICAL STEP Slowly remove at a 45 • angle to increase accuracy; 11. With one phase remaining, fill tube with cold D-PBS to a final volume of 10 mL; 12. Gently invert three times; 13. Centrifuge at 1000× g for 10 min at 4 • C. Aspirate remaining supernatant completely; 14. Prepare 1X Red Blood Cell Removal Solution (see Table 3); 15. Resuspend pellet in 500 µL of cold 1X Red Blood Cell Removal Solution, and incubate for 10 min at 4 • C; 16. Add 5 mL of PB buffer and centrifuge at 300× g for 10 min at 4 • C. Remove remaining supernatant completely and proceed to microglial magnetic beads labelling.
CRITICAL STEP Use the top end of a syringe plunger to mash and fully dissolve sample through strainer; CRITICAL STEP Transfer samples from 50 mL conical tubes to new 15 mL conical tubes after filtration; 5. Centrifuge samples at 300× g for 10 min at 4 °C. Remove supernatant completely; 6. Resuspend cell pellet with 1550 µL of cold D-PBS in the 15 mL conical tube; 7. Add 450 µL of debris removal solution and mix well; 8. Gently overlay 2 mL of D-PBS into tube. Be careful not to mix phases; OPTIONAL STEP Slowly pipette D-PBS with the conical tube at a 45° angle to prevent phase mixing; 9. Centrifuge samples at 300× g for 10 min at 4 °C; 10. After three phases are formed, aspirate the top two phases gently and completely discard; CRITICAL STEP Slowly remove at a 45° angle to increase accuracy; 11. With one phase remaining, fill tube with cold D-PBS to a final volume of 10 mL; 12. Gently invert three times; 13. Centrifuge at 1000× g for 10 min at 4 °C. Aspirate remaining supernatant completely; 14. Prepare 1X Red Blood Cell Removal Solution (see Table 3); 15. Resuspend pellet in 500 µL of cold 1X Red Blood Cell Removal Solution, and incubate for 10 min at 4 °C; 16. Add 5 mL of PB buffer and centrifuge at 300× g for 10 min at 4 °C. Remove remaining supernatant completely and proceed to microglial magnetic beads labelling. Figure 3. Schematic workflow of enzymatic digestion, centrifugation, and gradient removal of cellular debris. Critical modifications to protocol, highlighted in red, to allow for processing of small amounts of dissected brain tissue <50 mg.

Isolation of Mouse Microglia (Figure 4)
Magnetic Beads Incubation (CD11b + microglia labeling): 1. Prepare PB buffer solution (see Table 3); 2. Resuspend pellet in 90 µL cold PB buffer by slowly pipetting up and down; 3. Add 10 µL CD11b (Microglia) MicroBeads, human and mouse mix well; 4. Incubate in the dark for 15 min at 4 °C; 5. Add 1 mL of cold PB buffer and mix well; 6. Centrifuge at 300× g for 5 min at 4 °C; 7. Remove supernatant and resuspend cells in 500 µL PB buffer; Figure 3. Schematic workflow of enzymatic digestion, centrifugation, and gradient removal of cellular debris. Critical modifications to protocol, highlighted in red, to allow for processing of small amounts of dissected brain tissue <50 mg.
Resuspend pellet in 90 µL cold PB buffer by slowly pipetting up and down; 3.
Incubate in the dark for 15 min at 4 • C; 5.
Add 1 mL of cold PB buffer and mix well; 6.
Remove supernatant and resuspend cells in 500 µL PB buffer; CRITICAL STEP Wait until column is completely empty before proceeding to next step; OPTIONAL STEP To remove air bubbles from column after 500 µL rinse, use a needle syringe to pop bubbles; 9. Prepare 5 mL flow through collection tubes underneath MS columns for negative flow through collection; 10. Apply the 500 µL of CD11b + labeled cells (from step 7 above) into MS column attached to magnetic separator; CRITICAL STEP To increase purity of microglia, apply negative flow through from each collection tube back into corresponding MS column for a second time; 11. Wash MS column with 500 µL PB buffer three times; 12. After wash is complete, remove 5 mL collection tubes (containing negative fraction); 13. Place 5 mL collection tubes (containing negative fraction) into a 15 mL falcon to allow for centrifugation; CRITICAL STEP 14. Centrifuge 15 mL falcon containing 5 mL collection tubes with negative flow though at 300× g for 10 min at 4 °C. Set aside for astrocyte labelling in Section 3.6; 15. Remove MS column containing CD11b + bound cells from the separator, place it on corresponding collection tube, add 1 mL PB buffer, and immediately flush out to elute CD11b + magnetically labeled cells with provided plunger; 16. Repeat step 14 with an additional 1 mL of PB buffer to increase yield; CRITICAL STEP 17. Proceed to RNA extraction of eluted Cd11b + microglia.
CRITICAL STEP Wait until column is completely empty before proceeding to next step; OPTIONAL STEP To remove air bubbles from column after 500 µL rinse, use a needle syringe to pop bubbles;

9.
Prepare 5 mL flow through collection tubes underneath MS columns for negative flow through collection; 10. Apply the 500 µL of CD11b + labeled cells (from step 7 above) into MS column attached to magnetic separator; CRITICAL STEP Wait until column is completely empty before proceeding to next step; OPTIONAL STEP To remove air bubbles from column after 500 µL rinse, use a needle syringe to pop bubbles; 9. Prepare 5 mL flow through collection tubes underneath MS columns for negative flow through collection; 10. Apply the 500 µL of CD11b + labeled cells (from step 7 above) into MS column attached to magnetic separator; CRITICAL STEP To increase purity of microglia, apply negative flow through from each collection tube back into corresponding MS column for a second time; 11. Wash MS column with 500 µL PB buffer three times; 12. After wash is complete, remove 5 mL collection tubes (containing negative fraction); 13. Place 5 mL collection tubes (containing negative fraction) into a 15 mL falcon to allow for centrifugation; CRITICAL STEP 14. Centrifuge 15 mL falcon containing 5 mL collection tubes with negative flow though at 300× g for 10 min at 4 °C. Set aside for astrocyte labelling in Section 3.6; 15. Remove MS column containing CD11b + bound cells from the separator, place it on corresponding collection tube, add 1 mL PB buffer, and immediately flush out to + CRITICAL STEP To increase purity of microglia, apply negative flow through from each collection tube back into corresponding MS column for a second time; 11. Wash MS column with 500 µL PB buffer three times; 12. After wash is complete, remove 5 mL collection tubes (containing negative fraction); 13. Place 5 mL collection tubes (containing negative fraction) into a 15 mL falcon to allow for centrifugation; step; OPTIONAL STEP To remove air bubbles from column after 500 µL rinse, use a needle syringe to pop bubbles; 9. Prepare 5 mL flow through collection tubes underneath MS columns for negative flow through collection; 10. Apply the 500 µL of CD11b + labeled cells (from step 7 above) into MS column attached to magnetic separator; CRITICAL STEP To increase purity of microglia, apply negative flow through from each collection tube back into corresponding MS column for a second time; 11. Wash MS column with 500 µL PB buffer three times; 12. After wash is complete, remove 5 mL collection tubes (containing negative fraction); 13. Place 5 mL collection tubes (containing negative fraction) into a 15 mL falcon to allow for centrifugation; CRITICAL STEP 14. Centrifuge 15 mL falcon containing 5 mL collection tubes with negative flow though at 300× g for 10 min at 4 °C. Set aside for astrocyte labelling in Section 3.6; 15. Remove MS column containing CD11b + bound cells from the separator, place it on corresponding collection tube, add 1 mL PB buffer, and immediately flush out to elute CD11b + magnetically labeled cells with provided plunger; 16. Repeat step 14 with an additional 1 mL of PB buffer to increase yield;

CRITICAL STEP
17. Proceed to RNA extraction of eluted Cd11b + microglia.

CRITICAL STEP
14. Centrifuge 15 mL falcon containing 5 mL collection tubes with negative flow though at 300× g for 10 min at 4 • C. Set aside for astrocyte labelling in Section 3.6; 15. Remove MS column containing CD11b + bound cells from the separator, place it on corresponding collection tube, add 1 mL PB buffer, and immediately flush out to elute CD11b + magnetically labeled cells with provided plunger; 16. Repeat step 14 with an additional 1 mL of PB buffer to increase yield;

CRITICAL STEP
17. Proceed to RNA extraction of eluted Cd11b + microglia.
10. Apply the 500 µL of CD11b + labeled cells (from step 7 above) into MS column attached to magnetic separator; CRITICAL STEP To increase purity of microglia, apply negative flow through from each collection tube back into corresponding MS column for a second time; 11. Wash MS column with 500 µL PB buffer three times; 12. After wash is complete, remove 5 mL collection tubes (containing negative fraction); 13. Place 5 mL collection tubes (containing negative fraction) into a 15 mL falcon to allow for centrifugation; CRITICAL STEP 14. Centrifuge 15 mL falcon containing 5 mL collection tubes with negative flow though at 300× g for 10 min at 4 °C. Set aside for astrocyte labelling in Section 3.6; 15. Remove MS column containing CD11b + bound cells from the separator, place it on corresponding collection tube, add 1 mL PB buffer, and immediately flush out to elute CD11b + magnetically labeled cells with provided plunger; 16. Repeat step 14 with an additional 1 mL of PB buffer to increase yield;

CRITICAL STEP
17. Proceed to RNA extraction of eluted Cd11b + microglia. Figure 4. Schematic workflow of magnetic labeling for the sequential isolation of microglia and astrocytes. Following brain dissociation, debris, and red blood cell removal, (A) the purified homogenate is incubated with magnetically labelled anti-CD11b microbeads. Homogenate is then twice filtered through MS columns whilst attached to magnet. CD11b positively selected cells remain in the column and are then eluted into a fresh tube for microglial specific RNA extraction. The negative flow-through is collected for further processing. (B) Washed sample in negative flow-through columns are placed in 15 mL falcons to allow for centrifugation of cell pellet. Following centrifugation, supernatant is removed and pellet is processed for astrocyte isolation. (C) Sample incubated with anti-ACSA-2 microbeads, before astrocyte isolation and elution as described in (A).

Isolation of Mouse Astrocytes (Figure 4)
Sample preparation: Using forceps, carefully remove 5 mL reagent tubes from 15 mL conical tubes that were centrifuged in 3.5, step 13 and aspirate supernatant completely.
Add 10 µL of FcR Blocking Reagent, mix well, and incubate in the dark for 10 min at 4 • C; Magnetic Separation with MS Columns for Microglia 8. Place MS columns in the magnetic field of MACS Separator and prepare column by rinsing through 500 µL PB buffer. Discard flow through; CRITICAL STEP Wait until column is completely empty before proceeding to next step; OPTIONAL STEP To remove air bubbles from column after 500 µL rinse, use a needle syringe to pop bubbles; 9. Prepare 5 mL flow through collection tubes underneath MS columns for negative flow through collection; 10. Apply the 500 µL of CD11b + labeled cells (from step 7 above) into MS column attached to magnetic separator; CRITICAL STEP To increase purity of microglia, apply negative flow through from each collection tube back into corresponding MS column for a second time; 11. Wash MS column with 500 µL PB buffer three times; 12. After wash is complete, remove 5 mL collection tubes (containing negative fraction); 13. Place 5 mL collection tubes (containing negative fraction) into a 15 mL falcon to allow for centrifugation; CRITICAL STEP 14. Centrifuge 15 mL falcon containing 5 mL collection tubes with negative flow though at 300× g for 10 min at 4 °C. Set aside for astrocyte labelling in Section 3.6; 15. Remove MS column containing CD11b + bound cells from the separator, place it on corresponding collection tube, add 1 mL PB buffer, and immediately flush out to elute CD11b + magnetically labeled cells with provided plunger; 16. Repeat step 14 with an additional 1 mL of PB buffer to increase yield;

CRITICAL STEP
17. Proceed to RNA extraction of eluted Cd11b + microglia.
CRITICAL STEP Wait until column is completely empty before proceeding to next step; OPTIONAL STEP To remove air bubbles from column after 500 µL rinse, use a needle syringe to pop bubbles;

7.
Prepare 5 mL flow through collection tubes underneath MS columns for negative flow through collection; 8.
Apply the 500 µL of ACSA2 + labeled cells (from step 5 above) into MS column attached to magnetic separator; Magnetic Separation with MS Columns for Microglia 8. Place MS columns in the magnetic field of MACS Separator and prepare column by rinsing through 500 µL PB buffer. Discard flow through; CRITICAL STEP Wait until column is completely empty before proceeding to next step; OPTIONAL STEP To remove air bubbles from column after 500 µL rinse, use a needle syringe to pop bubbles; 9. Prepare 5 mL flow through collection tubes underneath MS columns for negative flow through collection; 10. Apply the 500 µL of CD11b + labeled cells (from step 7 above) into MS column attached to magnetic separator; CRITICAL STEP To increase purity of microglia, apply negative flow through from each collection tube back into corresponding MS column for a second time; 11. Wash MS column with 500 µL PB buffer three times; 12. After wash is complete, remove 5 mL collection tubes (containing negative fraction); 13. Place 5 mL collection tubes (containing negative fraction) into a 15 mL falcon to allow for centrifugation; CRITICAL STEP 14. Centrifuge 15 mL falcon containing 5 mL collection tubes with negative flow though at 300× g for 10 min at 4 °C. Set aside for astrocyte labelling in Section 3.6; 15. Remove MS column containing CD11b + bound cells from the separator, place it on corresponding collection tube, add 1 mL PB buffer, and immediately flush out to elute CD11b + magnetically labeled cells with provided plunger; 16. Repeat step 14 with an additional 1 mL of PB buffer to increase yield;

CRITICAL STEP
17. Proceed to RNA extraction of eluted Cd11b + microglia.

CRITICAL STEP
To increase purity of astrocytes, apply negative selection back through MS column a second time; OPTIONAL STEP To remove air bubbles from column entry disruption, use a needle syringe to remove obstructions;

9.
Wash MS column with 500 µL PB buffer three times; 10. Remove MS column containing ACSA2 + bound cells from the separator, place it on corresponding collection tube, add 1 mL AstroMACS Separation buffer, and immediately flush out to elute ACSA2 + magnetically labeled cells with provided plunger. 11. Repeat step 11 with an additional 1 mL of AstroMACS separation buffer to increase yield; Methods Protoc. 2022, 5, x FOR PEER REVIEW 7 of 16 Magnetic Separation with MS Columns for Microglia 8. Place MS columns in the magnetic field of MACS Separator and prepare column by rinsing through 500 µL PB buffer. Discard flow through; CRITICAL STEP Wait until column is completely empty before proceeding to next step; OPTIONAL STEP To remove air bubbles from column after 500 µL rinse, use a needle syringe to pop bubbles; 9. Prepare 5 mL flow through collection tubes underneath MS columns for negative flow through collection; 10. Apply the 500 µL of CD11b + labeled cells (from step 7 above) into MS column attached to magnetic separator; CRITICAL STEP To increase purity of microglia, apply negative flow through from each collection tube back into corresponding MS column for a second time; 11. Wash MS column with 500 µL PB buffer three times; 12. After wash is complete, remove 5 mL collection tubes (containing negative fraction); 13. Place 5 mL collection tubes (containing negative fraction) into a 15 mL falcon to allow for centrifugation; CRITICAL STEP 14. Centrifuge 15 mL falcon containing 5 mL collection tubes with negative flow though at 300× g for 10 min at 4 °C. Set aside for astrocyte labelling in Section 3.6; 15. Remove MS column containing CD11b + bound cells from the separator, place it on corresponding collection tube, add 1 mL PB buffer, and immediately flush out to elute CD11b + magnetically labeled cells with provided plunger; 16. Repeat step 14 with an additional 1 mL of PB buffer to increase yield;

CRITICAL STEP
17. Proceed to RNA extraction of eluted Cd11b + microglia.

CRITICAL STEP CRITICAL STEP
12. Proceed to RNA extraction of eluted ACSA2 + astrocytes.

RNA Extraction from Microglial and Astrocyte Samples (Figures 5-7)
Preparation of samples for RNA Extraction
Add 500 µL TRIzol, sonicate pellet completely, and transfer solution to 1.5 mL Eppendorf tubes. Proceed with subsequent RNA extraction. Samples can be stored at −20 • C for later extraction;
Add 200 µL chloroform, thoroughly mix by shaking, and incubate at RT for 2 min. 5.
The mixture separates into 3 layers which include a colorless upper phase, white interphase, and pink phenol-chloroform lower phase; Methods Protoc. 2022, 5, x FOR PEER REVIEW 7 of 16 Magnetic Separation with MS Columns for Microglia 8. Place MS columns in the magnetic field of MACS Separator and prepare column by rinsing through 500 µL PB buffer. Discard flow through; CRITICAL STEP Wait until column is completely empty before proceeding to next step; OPTIONAL STEP To remove air bubbles from column after 500 µL rinse, use a needle syringe to pop bubbles; 9. Prepare 5 mL flow through collection tubes underneath MS columns for negative flow through collection; 10. Apply the 500 µL of CD11b + labeled cells (from step 7 above) into MS column attached to magnetic separator; CRITICAL STEP To increase purity of microglia, apply negative flow through from each collection tube back into corresponding MS column for a second time; 11. Wash MS column with 500 µL PB buffer three times; 12. After wash is complete, remove 5 mL collection tubes (containing negative fraction); 13. Place 5 mL collection tubes (containing negative fraction) into a 15 mL falcon to allow for centrifugation; CRITICAL STEP 14. Centrifuge 15 mL falcon containing 5 mL collection tubes with negative flow though at 300× g for 10 min at 4 °C. Set aside for astrocyte labelling in Section 3.6; 15. Remove MS column containing CD11b + bound cells from the separator, place it on corresponding collection tube, add 1 mL PB buffer, and immediately flush out to elute CD11b + magnetically labeled cells with provided plunger; 16. Repeat step 14 with an additional 1 mL of PB buffer to increase yield; CRITICAL STEP In a 45 • angle, being careful not to mix phases together, remove clear top layer containing RNA into a new tube. For optimal purity, closely view tube for phase movement and do not transfer contaminated mixed-phase solution ( Figure 5); OPTIONAL STEP Should any of the three layers be accidentally mixed, centrifuge samples again at 4 • C for 15 min at 12,000× g and repeat the upper-phase extraction, which contains RNA ( Figure 5); Methods Protoc. 2022, 5, 77 9 of 15 CRITICAL STEP In a 45° angle, being careful not to mix phases together, remove clear top layer containing RNA into a new tube. For optimal purity, closely view tube for phase movement and do not transfer contaminated mixed-phase solution ( Figure 5); OPTIONAL STEP Should any of the three layers be accidentally mixed, centrifuge samples again at 4 °C for 15 min at 12,000× g and repeat the upper-phase extraction, which contains RNA ( Figure 5); 7. Add 500 µL of 100% isopropanol to each sample, invert tubes three times, and incubate in 4 °C for 10 min; 8. Centrifuge in 4 °C for 10 min. Tubes should be spun with the hinged cap side up, which will be used as a physical guideline for identifying pellets at later stages. It is important to maintain this tube angle in all centrifugation steps; 9. Due to use of a 45° rotor, a small, white translucent, gel-like pellet should appear after centrifugation along the bottom of the tube on the same side of the cap's hinge. Carefully discard supernatant while maintaining pellet in the tube; CRITICAL STEP It is recommended to grade down pipette sizes for increased precision of supernatant removal; OPTIONAL STEP For smaller samples, it is recommended to perform procedure alongside a larger tissue sample (50-100 mg) as a reference control for pellet location;
Centrifuge in 4 • C for 10 min. Tubes should be spun with the hinged cap side up, which will be used as a physical guideline for identifying pellets at later stages. It is important to maintain this tube angle in all centrifugation steps; 9.
Due to use of a 45 • rotor, a small, white translucent, gel-like pellet should appear after centrifugation along the bottom of the tube on the same side of the cap's hinge. Carefully discard supernatant while maintaining pellet in the tube; Methods Protoc. 2022, 5, x FOR PEER REVIEW 7 of 16 Magnetic Separation with MS Columns for Microglia 8. Place MS columns in the magnetic field of MACS Separator and prepare column by rinsing through 500 µL PB buffer. Discard flow through; CRITICAL STEP Wait until column is completely empty before proceeding to next step; OPTIONAL STEP To remove air bubbles from column after 500 µL rinse, use a needle syringe to pop bubbles; 9. Prepare 5 mL flow through collection tubes underneath MS columns for negative flow through collection; 10. Apply the 500 µL of CD11b + labeled cells (from step 7 above) into MS column attached to magnetic separator; CRITICAL STEP To increase purity of microglia, apply negative flow through from each collection tube back into corresponding MS column for a second time; 11. Wash MS column with 500 µL PB buffer three times; 12. After wash is complete, remove 5 mL collection tubes (containing negative fraction); 13. Place 5 mL collection tubes (containing negative fraction) into a 15 mL falcon to allow for centrifugation; CRITICAL STEP 14. Centrifuge 15 mL falcon containing 5 mL collection tubes with negative flow though at 300× g for 10 min at 4 °C. Set aside for astrocyte labelling in Section 3.6; 15. Remove MS column containing CD11b + bound cells from the separator, place it on corresponding collection tube, add 1 mL PB buffer, and immediately flush out to elute CD11b + magnetically labeled cells with provided plunger; 16. Repeat step 14 with an additional 1 mL of PB buffer to increase yield;

CRITICAL STEP
17. Proceed to RNA extraction of eluted Cd11b + microglia.
CRITICAL STEP It is recommended to grade down pipette sizes for increased precision of supernatant removal; OPTIONAL STEP For smaller samples, it is recommended to perform procedure alongside a larger tissue sample (50-100 mg) as a reference control for pellet location; 10. Resuspend pellet with 1 mL of 75% ethanol, vortex, and centrifuge in 4 °C for 5 min at 7500× g; 11. A thin gel-like pellet should appear after centrifugation in the same 45° location. Discard as much supernatant as possible while maintaining visible sight of pellet. If pellet is not clearly visible, use a larger tissue sample for reference of location; CRITICAL STEP For optimal purity, leave a small amount of supernatant (~ 20 µL) in the tube and centrifuge at 7500× g in 4 °C for 5 min again. After spin, discard supernatant with P2 or P10 pipette; CRITICAL STEP It is recommended to grade down pipette sizes for increased precision of supernatant removal and preservation of RNA;  Magnetic Separation with MS Columns for Microglia 8. Place MS columns in the magnetic field of MACS Separator and prepare column by rinsing through 500 µL PB buffer. Discard flow through; CRITICAL STEP Wait until column is completely empty before proceeding to next step; OPTIONAL STEP To remove air bubbles from column after 500 µL rinse, use a needle syringe to pop bubbles; 9. Prepare 5 mL flow through collection tubes underneath MS columns for negative flow through collection; 10. Apply the 500 µL of CD11b + labeled cells (from step 7 above) into MS column attached to magnetic separator; CRITICAL STEP To increase purity of microglia, apply negative flow through from each collection tube back into corresponding MS column for a second time; 11. Wash MS column with 500 µL PB buffer three times; 12. After wash is complete, remove 5 mL collection tubes (containing negative fraction); 13. Place 5 mL collection tubes (containing negative fraction) into a 15 mL falcon to allow for centrifugation; CRITICAL STEP 14. Centrifuge 15 mL falcon containing 5 mL collection tubes with negative flow though at 300× g for 10 min at 4 °C. Set aside for astrocyte labelling in Section 3.6; 15. Remove MS column containing CD11b + bound cells from the separator, place it on corresponding collection tube, add 1 mL PB buffer, and immediately flush out to elute CD11b + magnetically labeled cells with provided plunger; 16. Repeat step 14 with an additional 1 mL of PB buffer to increase yield; CRITICAL STEP 17. Proceed to RNA extraction of eluted Cd11b + microglia.
CRITICAL STEP For optimal purity, leave a small amount of supernatant (~20 µL) in the tube and centrifuge at 7500× g in 4 • C for 5 min again. After spin, discard supernatant with P2 or P10 pipette;

Expected Results and Discussion
In many experimental paradigms, the ability to isolate multiple CNS cell types from a single homogenate is advantageous. It allows for exploration of cellular morphology with the relatively gently nature of MACs isolations preserving cell integrity and networks of processes [20,21]. Isolated microglia exhibit a typical longitudinal bipolar cell body with ramified pseudopodia, while astrocytes display branched cellular processes and arborizations that allow for extra somatic proteins to be visualized [13,17,22]. Further adding to the advantageous nature of this technology, is its high throughput capacity in a time and cost-effective manner. Seminal results show isolated microglia and astrocytes display high purity (94% and 96%, respectively), with a total number of 4 × 10 5 astrocytes and 4.2 × 10 5 microglia obtained from one adult mouse brain each [23]. Optimization of protocols have seen these numbers refined, with microglial cell counts of 50,000-200,000 from bilateral hippocampus [24] and single hemispheres [25]. Viability of microglia and astrocytes separated by MACS are routinely observed to be greater than 90% [17,23]. Of relevance to this study, MACs isolations can also be used to perform multi-omic analyses of subpopulations of immune cells, enhancing our understanding of complex cellular networks.
Since RNA sequencing was first introduced in 2008 [26,27], it has become a common tool in molecular biology, influencing almost every aspect of our understanding of genomic function. Although outside the methodological scope of this manuscript, the standard workflow consists of RNA extraction followed by mRNA enrichment or ribosomal RNA depletion. Subsequently, cDNA synthesis and creation of an adaptor-ligated sequencing library is performed before libraries are sequenced on a high-throughput machine platform to a read depth of 10-30 million reads per sample. The final steps are bioinformatic based, aligning sequencing reads to the transcriptome of the species of interest, before normalizing samples and investigating levels of differential gene expression (reviewed in [28]). Indeed, to date, almost 100 distinct methods have been derived from the standard RNA-seq protocol [29]. This combined with the affordability and accessibility of sequencing platforms provided by commercially available pharmaceutical/biotech companies, as well as academic shared-resources, means that RNA sequencing has now emerged as a commonplace technique in experimental designs across research fields.
Here, we describe an application of immuno-magnetic cell separation that allows for the sequential isolation of glial cell populations (astrocytes and microglia) from the adult mouse brain for downstream transcriptomic analysis. This technique is cost-effective and modifiable for the study of multiple responses within the biological context of a single mouse brain microenvironment. It is also advantageous in terms of reducing the number of mice required to assess cellular responses in models with limited tissue for analysis (e.g., ipsilateral brain injury).
Using this sequential isolation protocol, we demonstrate the ability to isolate sufficient quantities of RNA suitable for downstream transcriptomic applications. Chip based capillary electrophoresis of RNA from isolated microglia and astrocytes shows high quality and minimal degradation as evidenced by high ratios of 18S to 28S ribosomal bands, and minimal baseline signal between peaks ( Figure 8B). Electropherogram visualization and integrity algorithms routinely classified samples as having integrity scores between 8-9, and total yields displaying concentrations of 200 ng/µL or higher ( Figure 8C). Of importance, low-quality RNA, can substantially affect the sequencing results (e.g., uneven gene coverage, 3 -5 transcript bias) and lead to erroneous biological conclusions [30]. We show that this protocol not only results in high quality RNA, but downstream transcriptomic control measures show high %Q-and Phred scores ( Figure 8D,E), evidencing high quality sequencing data (99.9% base call accuracy), with low rates of false positive gene expression.  Advancements in sequencing-based technologies and comparative transcriptomic studies have generated useful resources for identifying the molecular signatures of many CNS cell types, including microglia and astrocytes [31][32][33]. As a result, we can use transcriptomic profiling to determine the composition of cell populations. In both young and aged isolated microglia, we confirm the elevated expression of microglial specific markers, indicating highly enriched microglial populations ( Figure 9A-D).
To achieve this result, the order of sequential isolation is of critical importance. Given Advancements in sequencing-based technologies and comparative transcriptomic studies have generated useful resources for identifying the molecular signatures of many CNS cell types, including microglia and astrocytes [31][32][33]. As a result, we can use transcriptomic profiling to determine the composition of cell populations. In both young and aged isolated microglia, we confirm the elevated expression of microglial specific markers, indicating highly enriched microglial populations ( Figure 9A-D).
Methods Protoc. 2022, 5, x FOR PEER REVIEW 13 of 16 Figure 9. Transcriptomic validation of microglia isolated from adult mouse brain. mRNA analysis shows isolated microglial populations have high expression of microglial specific markers (A) Aif1, (B) Cx3cr1, (C) Itgam1 and (D) Tmem119 (young microglia n = 9, young astrocyte n = 9, aged microglia n = 10, aged astrocyte n = 10). Data from 3 independent experiments run on different days, a total of n = 3/day for young mice and n = 3, 3, 4 per day for aged mice.
Following Cd11b + microglial selection, the negative flow-through is collected and used for subsequent isolation of astrocytes. This negative collection is gently centrifuged before astrocytes are isolated using ASCA2 + microbeads selection. Similar to prior microglial preparations, incubated ASCA2 + volumes are passed through columns twice to increase yield through positive selection magnetic binding. We confirmed this sequential step of the protocol is specific for astrocyte populations, evidenced by the expression of elevated astrocyte markers Gfap, Atp1b2, Slc1a2 and Slc1a3 in both young and aged astrocytes, but not in microglial preparations ( Figure 10A-D). In addition to confirming positive selection of astrocytes using this method, we also show that isolated young and aged astrocytes display elevated levels of Atp1b2, in agreement with a previous study identifying ATP1B2 as the target epitope of ASCA2 antibody binding [14]. . Transcriptomic validation of microglia isolated from adult mouse brain. mRNA analysis shows isolated microglial populations have high expression of microglial specific markers (A) Aif1, (B) Cx3cr1, (C) Itgam1 and (D) Tmem119 (young microglia n = 9, young astrocyte n = 9, aged microglia n = 10, aged astrocyte n = 10). Data from 3 independent experiments run on different days, a total of n = 3/day for young mice and n = 3, 3, 4 per day for aged mice.
To achieve this result, the order of sequential isolation is of critical importance. Given microglia constitute only 5-10% of total brain cells [5], Cd11b + magnetic labelling and selection should occur first to provide the highest probably of cell capture at sufficient yields. In addition, we pass the Cd11b + cell homogenate through the MS filter columns twice before the elution of positively selected cells. We believe this step is critical to increase the purity and yield of Cd11b + microglial cells selected, especially given our preparations are from a relatively small starting amount of tissue (singular micro punch <50 mg, as would be used in traumatic brain injury models of ipsilateral injury [34]).
Following Cd11b + microglial selection, the negative flow-through is collected and used for subsequent isolation of astrocytes. This negative collection is gently centrifuged before astrocytes are isolated using ASCA2 + microbeads selection. Similar to prior microglial preparations, incubated ASCA2 + volumes are passed through columns twice to increase yield through positive selection magnetic binding. We confirmed this sequential step of the protocol is specific for astrocyte populations, evidenced by the expression of elevated astrocyte markers Gfap, Atp1b2, Slc1a2 and Slc1a3 in both young and aged astrocytes, but not in microglial preparations ( Figure 10A-D). In addition to confirming positive selection of astrocytes using this method, we also show that isolated young and aged astrocytes display elevated levels of Atp1b2, in agreement with a previous study identifying ATP1B2 as the target epitope of ASCA2 antibody binding [14]. Figure 10. Transcriptomic validation of sequentially isolated astrocytes from adult mouse brain. mRNA analysis shows astrocytes that are isolated after microglial preparations have high expression of astrocyte specific markers (A) Gfap, (B) Atp1b2, (C) Slc1a2 and (D) Slc1a3 (young microglia n = 9, young astrocyte n = 9, aged microglia n = 10, aged astrocyte n = 10). Data from 3 independent experiments run on different days, a total of n = 3/day for young mice and n = 3, 3, 4 per day for aged mice.

Conclusions
Our modified magnetic cell sorting technique allows for sequential isolation of microglia and astrocytes that is effective in both the young and aged adult mouse brain. This protocol allows for the cost-effective assessment of cellular interactions in the biological context of the individual mouse brain, providing a powerful tool for the holistic analysis of CNS responses. We show that in our experience, a single mouse cortex and/or hippocampus is sufficient to isolate cells for downstream transcriptomic analysis, allowing for the ethical reduction and refinement of the experimental animals required. Figure 10. Transcriptomic validation of sequentially isolated astrocytes from adult mouse brain. mRNA analysis shows astrocytes that are isolated after microglial preparations have high expression of astrocyte specific markers (A) Gfap, (B) Atp1b2, (C) Slc1a2 and (D) Slc1a3 (young microglia n = 9, young astrocyte n = 9, aged microglia n = 10, aged astrocyte n = 10). Data from 3 independent experiments run on different days, a total of n = 3/day for young mice and n = 3, 3, 4 per day for aged mice.

Conclusions
Our modified magnetic cell sorting technique allows for sequential isolation of microglia and astrocytes that is effective in both the young and aged adult mouse brain. This protocol allows for the cost-effective assessment of cellular interactions in the biological context of the individual mouse brain, providing a powerful tool for the holistic analysis of CNS responses. We show that in our experience, a single mouse cortex and/or hippocampus is sufficient to isolate cells for downstream transcriptomic analysis, allowing for the ethical reduction and refinement of the experimental animals required.