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Flow cytometry

Ua Uri T abori
SS Simone C Stone

Last updated date: Jun 28, 2022 Views: 531 Forks: 0

original An abbreviated version of this protocol was published in Nat Med in Jan, 2022
Genomic predictors of response to PD-1 inhibition in children with germline DNA replication repair deficiency
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  1. Add single cell suspension in 2% FCS PBS (FACS buffer) to a 96-well round bottom plate.
  2. Centrifuge at 1500 rpm for 2 minutes at room temperature.
  3. Invert plate to remove FACS buffer.
  4. Add 25uL of Fc Block (1:200, BD) per sample.
  5. Incubate for 10 minutes at room temperature.
  6. Prepare 2x stains for each sample. 25uL of a 2x stain (including antibody mix and viability dye) is added directly onto the Fc Block reagent (from step 4) to create a 1x stain.
  7. Add 25uL of 2x surface stain to each sample and incubate for 30 minutes at 4*C.
  8. To wash cells, first add 150uL of FACS buffer onto the stain (wash #1) and spin at 1500 rpm at room temperature for 2 minutes.
  9. Remove supernatant, add 200uL of FACS buffer, and gently mix with the pipette (wash #2) and spin at 1500 rpm at room temperature for 2 minutes. Remove supernatant.
  10. Fix cells in 50uL of eBioscience FoxP3 fixation/permeabilization buffer (1 part fixation buffer: 3 parts diluent) for 30 minutes at 4*C
  11. Wash cells as steps 8 and 9 (except, adjust spinning to 1800 rpm after fixation for this and the following steps). If intracellular staining is required wash cells with 1X eBioscience permwash buffer instead.
  12. If intracellular staining is required:
    1. After washing cells with 1X eBioscience permwash buffer, spin and remove supernatant.
    2. Add 50uL of antibody mix in 1X eBioscience permwash buffer per plate well.
    3. Stain cells for 60 minutes at 4*C.
  13. Wash cells as steps 8 and 9. 
  14. Remove supernatant and resuspend in FACS buffer.
  15. Transfer to a FACS tube and protect from light until ready to run the samples.
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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. abori, U and Stone, S C(2022). Flow cytometry. Bio-protocol Preprint. bio-protocol.org/prep1752.
  2. Das, A., Sudhaman, S., Morgenstern, D., Coblentz, A., Chung, J., Stone, S. C., Alsafwani, N., Liu, Z. A., Karsaneh, O. A. A., Soleimani, S., Ladany, H., Chen, D., Zatzman, M., Cabric, V., Nobre, L., Bianchi, V., Edwards, M., Nahum, L. C. S., Ercan, A. B., Nabbi, A., Constantini, S., Dvir, R., Yalon-Oren, M., Campino, G. A., Caspi, S., Larouche, V., Reddy, A., Osborn, M., Mason, G., Lindhorst, S., Bronsema, A., Magimairajan, V., Opocher, E., Mola, R. L. D., Sabel, M., Frojd, C., Sumerauer, D., Samuel, D., Cole, K., Chiaravalli, S., Massimino, M., Tomboc, P., Ziegler, D. S., George, B., Damme, A. V., Hijiya, N., Gass, D., McGee, R. B., Mordechai, O. and Bowers, D. C.(2022). Genomic predictors of response to PD-1 inhibition in children with germline DNA replication repair deficiency. Nat Med 28(1). DOI: 10.1038/s41591-021-01581-6

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