Improve Research Reproducibility A Bio-protocol resource
bpdetail_icon_lock
Preprint

Flow cytometry

TS Takuya Sekine
AP André Perez-Potti
MB Marcus Buggert

Last updated date: Feb 11, 2021 Views: 933 Forks: 0

original An abbreviated version of this protocol was published in Science Immunology in Jul, 2020
TOX is expressed by exhausted and polyfunctional human effector memory CD8+ T cells
download pdf Download PDF
ask Ask a question
cite How to cite
nfavorite Favorite
  1. Resuspended PBMCs at 106 cells/ml and distribute in a V-bottom 96-well plate at 100ul per well. 
  2. Wash cells by adding 150 ul of fluorescence-activated cell sorting (FACS) buffer [phosphate-buffered saline (PBS) supplemented with 2% fetal bovine serum and 2 mM EDTA] and spin down for 3 minutes. Discard supernatant without disrupting cell pellet.
  3. Add 1ul of SA-BV421 or SA-PE conjugated MHC class I tetramer (25ug/ml) directly to the cell pellet.  Gently vortex to ensure cell pellet is resuspended.  Incubate for 20minutes at room temperature in the dark. Skip to step 4 if not relevant. 
  4. Add 1ul of CCR7 APC-Cy7 antibody (clone G043H7, BioLegend) directly to the cell pellet. Vortex gently to ensure cell pellet has disaggregated.  Incubate for 10 min at 37°C.
  5. Add 50 ul of the surface antibody cocktail reconstituted in FACS buffer to each well.  
  6. Incubate 20 min at room temperature in the dark.
  7. Add 150ul FACS buffer and spin down at 300rpm for 3 minutes. Discard supernatant without disrupting cell pellet.
  8. Add 100ul 1XFixation/Permeablization reagent (FoxP3/ Transcription Factor Staining Buffer Set - eBioscience) to the cell pellet and resuspend well. Cells were fixed/permeabilized for 20 min at room temperature in the dark.
  9. Wash once by adding 150ul 1X Perm/Wash Buffer and spin down at 400rpm for 3 minutes.  Discard supernatant without disrupting cell pellet
  10. Add 50 ul of the Intracellular antibody cocktail resuspended in 1X Perm/Wash buffer.
  11. Incubate for 1 hour at room temperature in the dark. 
  12. Wash cells with 1X Perm/Wash buffer.
  13. Resuspend cells in 1% paraformaldehyde (Biotium).  Immediately proceed toFACS analysis or store at 4°C and analyse within 24 hours.
Copyright: Content may be subjected to copyright.
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Sekine, T, Perez-Potti, A and Buggert, M(2021). Flow cytometry. Bio-protocol Preprint. bio-protocol.org/prep835.
  2. Sekine, T., Perez-Potti, A., Nguyen, S., Gorin, J., Wu, V. H., Gostick, E., Llewellyn-Lacey, S., Hammer, Q., Falck-Jones, S., Vangeti, S., Yu, M., Smed-Sörensen, A., Gaballa, A., Uhlin, M., Sandberg, J. K., Brander, C., Nowak, P., Goepfert, P. A., Price, D. A., Betts, M. R. and Buggert, M.(2020). TOX is expressed by exhausted and polyfunctional human effector memory CD8+ T cells . Science Immunology 5(49). DOI: 10.1126/sciimmunol.aba7918

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

0/150

tip Tips for asking effective questions

+ Description

Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.

post Post a Question
0 Q&A
This protocol preprint was submitted via the "Request a Protocol" track.