Amplification of Signal on Cell Surfaces in Molecular Cascades
Abstract
:1. Introduction
2. Results and Discussion
2.1. Design Considerations for Amplification Cascade Reaction
2.2. AMPLIFY(YESCD19YESCD45) Cascade Reaction
2.3. AMPLIFY(YESCD45RAYESCD3) Cascade Reaction
3. Conclusions
4. Materials and Methods
4.1. Materials
- 0 =/5IAbRQ/TACGTATCGAGGCTTTTACTTCTATCTCTTTCTT;
- 1 = GAAAGTGTTAAAGAAAGAGATAGAAGTAAAAGCCTCGATACGTA;
- 1(Cy5) = GAAAGTGTTAAAGAAAGAGATAGAAGTAAAAGCCTCGATAC
- GTA/3Cy5Sp/;
- 2 =/5ThioMC6-D/GGCTTTTACTTCTATCTCTTTCTTTAACACTTTCCTATACTT
- ATGCACTT;
- 3 = GAAAGTGTTAAAGAAAGAGATTGAAGTAAATGCCTC;
- 4 =/5ThioMC6-D/CTTTACGATTTGGTTACGTATCGAGGCATTTACTTCAATCT
- CTTTCTT;
- 5 =/5BioTinTEG/AGTAAATGCCTCGATACGTAACCAAATCGTTAAGCC/36-FAM/;
- 6 =/5IABkFQ/GGCTTAACGATTTGGTTACGTATCGA.
4.2. Methods
- PBMCs (25 × 106/mL) were pre-incubated with anti-CD32 antibodies (12.5 ng/mL, Clone IV.3, STEMCELL Technologies, Vancouver, BC, Canada) and Shredded Salmon Sperm DNA (125 ug/mL, Ambion, Austin, TX, USA) in buffer (PBS supplemented with 0.5% (w/v) BSA and 2 mM EDTA) at room temperature for 10 min.
- The conjugates of antibodies participated in the cascade reaction together with the corresponding Pacific Blue conjugated antibody were added to PBMCs in an amount that was determined after previous titration, to be sure that all antigens on the cell surface would be saturated with minimal non-specific binding registered. The mixture was incubated on ice for 30 min with periodic gentle mixing.
- Samples were washed twice with up to 15 mL buffer by centrifugation (350 g × 10 min, at 5 °C).
- The final pellet was resuspended to obtain a concentration of cells of 50 × 106 PBMCs per ml of buffer at room temperature.
- About 5 × 106 PBMCs (100 µL) were resuspended in a final volume of 500 µL in a 5 mL tube for flow cytometry and the sample subjected to flow cytometry at a rate 12 µL/min. After the monitoring of the baseline during the first 100 s, the duplex F-5∗6 was added to sample to reach a final concentration of about 700 nM; after the second 100 s, the strand 0 was added to reach a final concentration about 200 nM. In some experiments, after the next 1100 s, strand 2 was added to obtain a final concentration of about 400 nM.
- PBMCs (25 × 106/mL) were pre-incubated with anti-CD32 antibodies (12.5 ng/mL, Clone IV.3, STEMCELL Technologies) and Shredded Salmon Sperm DNA (125 ug/mL, Ambion) in PBS supplemented with 0.5% (w/v) BSA and 2 mM EDTA (Buffer) at room temperature for 10 min.
- The CD45-(1∗4) conjugate was added to PBMCs in an amount that was determined after previous titration, to be sure that all antigens on the cell surface would be saturated, with minimal non-specific binding registered. The mixture was incubated on ice for 30 min with periodic gentle mixing.
- Samples were washed twice with up to 15 mL of buffer by centrifugation (350 g × 10 min, at 5 °C).
- Cells were resuspended in a concentration of 25 × 106/mL in buffer and strand 2 was added to obtain a final concentration 400 nM with subsequent incubation at room temperature for 30 min.
- Samples were washed twice with up to 15 mL of buffer by centrifugation (350 g × 10 min, at 5 °C).
- Cells were resuspended at a concentration of 25 × 106/mL in buffer and strand 3 was added to obtain a final concentration of 400 nM with subsequent incubation on ice for 30 min.
- Samples were washed twice with up to 15 mL buffer by centrifugation (350 g × 10 min, at 5 °C).
- The CD19-(1∗2) conjugate together with Pacific Blue anti-CD20 antibodies was added to PBMCs in an amount that was determined after previous titration, to be sure that all antigens on the cell surface would be saturated with minimal non-specific binding registered. The mixture was incubated on ice for 30 min with periodic gentle mixing.
- Samples were washed twice with up to 15 mL of buffer by centrifugation (350 g × 10 min, at 5 °C).
- The final pellet was resuspended to obtain a concentration of cells at 50 × 106 PBMCs per ml of buffer at room temperature.
- About 5 × 106 PBMCs (100 µL) were resuspended in a final volume 500 µL in a 5 mL tube for flow cytometry and the sample subjected to flow cytometry at a rate of 12 µL/min. After the monitoring of the baseline during the first 100 s, the duplex F-5∗6 was added to the sample to obtain a final concentration of about 700 nM; after the second 100 s, the strand 0 was added to obtain a final concentration of about 200 nM. After the next 1100 s, strand 2 was added to obtain final concentration of about 400 nM.
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Rudchenko, S.; Taylor, S.; Milosavic, N.; Rudchenko, M.; Wedderhoff Tissi, B.; Mapara, M.Y.; Stojanovic, M.N. Amplification of Signal on Cell Surfaces in Molecular Cascades. Cells 2023, 12, 2858. https://doi.org/10.3390/cells12242858
Rudchenko S, Taylor S, Milosavic N, Rudchenko M, Wedderhoff Tissi B, Mapara MY, Stojanovic MN. Amplification of Signal on Cell Surfaces in Molecular Cascades. Cells. 2023; 12(24):2858. https://doi.org/10.3390/cells12242858
Chicago/Turabian StyleRudchenko, Sergei, Steven Taylor, Nenad Milosavic, Maria Rudchenko, Betina Wedderhoff Tissi, Markus Y. Mapara, and Milan N. Stojanovic. 2023. "Amplification of Signal on Cell Surfaces in Molecular Cascades" Cells 12, no. 24: 2858. https://doi.org/10.3390/cells12242858
APA StyleRudchenko, S., Taylor, S., Milosavic, N., Rudchenko, M., Wedderhoff Tissi, B., Mapara, M. Y., & Stojanovic, M. N. (2023). Amplification of Signal on Cell Surfaces in Molecular Cascades. Cells, 12(24), 2858. https://doi.org/10.3390/cells12242858