A Simple Protocol for Isolating Hemolymph from Single Drosophila melanogaster Adult Flies
Abstract
:1. Introduction
2. Experimental Design
2.1. Materials
2.2. Equipment
2.3. Statistical Analysis
3. Procedure
- Prepare the nanoinjector per the manufacturer’s instructions and attach a glass capillary filled with mineral oil. Set the injector to “empty” to remove excess oil from the needle, and then fill the capillary needle with 5 μL of the diluted 2.5× protease inhibitor (PI) in 0.1 M of the sodium phosphate buffer (pH 7.4) per the manufacturer’s protocol. The flow rate is set at 69 nL/s and the fill rate at 30 nL/s.
- Group Collection: Anesthetize the flies under carbon dioxide and inject individuals with 100 nL of PI. After all individuals are injected, set the nanoinjector to the “fill” setting.
- CRITICAL STEP TIP: Before withdrawing hemolymph from the flies, there should be PI left in the needle (approximately 10 nL) to come into contact with the extracted hemolymph and preserve it.
- Reinsert the capillary into the mesothorax region of the adult fly and slightly pulse the nanoinjector for one to two seconds, creating a small vacuum allowing the hemolymph to freely draw upward into the needle.
- Once hemolymph is extracted from all individuals and the contents of the needle are emptied into a 1.5 mL microcentrifuge tube containing 20 μL of 2.5× PI on ice.
- OPTIONAL STEP: If extracting hemolymph from a large number of flies, periodically empty the collected hemolymph (approximately every 5–10 individuals) into the microcentrifuge tube on ice to preserve the hemolymph and repeat until all individuals are processed.
- Individual Collection: While flies are anesthetized under CO2, prepare the nanoinjector similarly to 3.1 but fill the capillary with 2.5 μL of PI instead of 5 μL. Inject a single individual with 100 nL of PI and then immediately reverse the flow of the nanoinjector to collect the hemolymph.
- Empty the contents of the needle into a 0.5 mL microcentrifuge tube containing 2.5 μL of 2.5× PI on ice.
- Repeat this process for all individuals in the trial.
- OPTIONAL STEP: You may change the needle in between individuals to avoid cross-contamination.
- Gently vortex the tube with extracted hemolymph and store it on ice until the hemocytes are ready for analysis.
- TROUBLESHOOTING: After creating the vacuum, you may need to slightly pull back on the needle in order for the hemolymph to flow backward into the needle.
4. Results and Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Jones, K.; Eleftherianos, I. A Simple Protocol for Isolating Hemolymph from Single Drosophila melanogaster Adult Flies. Methods Protoc. 2023, 6, 100. https://doi.org/10.3390/mps6050100
Jones K, Eleftherianos I. A Simple Protocol for Isolating Hemolymph from Single Drosophila melanogaster Adult Flies. Methods and Protocols. 2023; 6(5):100. https://doi.org/10.3390/mps6050100
Chicago/Turabian StyleJones, Kirah, and Ioannis Eleftherianos. 2023. "A Simple Protocol for Isolating Hemolymph from Single Drosophila melanogaster Adult Flies" Methods and Protocols 6, no. 5: 100. https://doi.org/10.3390/mps6050100
APA StyleJones, K., & Eleftherianos, I. (2023). A Simple Protocol for Isolating Hemolymph from Single Drosophila melanogaster Adult Flies. Methods and Protocols, 6(5), 100. https://doi.org/10.3390/mps6050100