Flow Cytometry ICS Nuclear Antigens
Michael Betts, Gregory Golden
Abstract
High-parameter flow cytometry enables identification and characterization of a wide range of cell populations within a biological sample. A combined analysis of extracellular epitope staining (ECS) and intra-cellular epitope staining (ICS) using a collection of fluorophore-labeled antibodies sufficiently identifies discrete cell populations and their respective phenotypes. Importantly, ECS/ICS can be applied to cryo-preserved cell suspensions recovered in tissue culture media, enabling samples to be conveniently analyzed after collection and proper cryopreservation. However, consistent cryopreserved sample recovery and ECS procedures are critical to data comparison across multiple experiments. Herein, we describe a standardized protocol for cryopreserved sample recovery and ECS/ICS procedures for cell-surface and intra-cellular epitope labeling.
Steps
Procedure
Thawing and Resting
a. Pre-warm R10 media in a 37°C water bath.
b. Thaw samples in-vial using a 37°C water bath.
c. Add thawed cells to 14mL of R10, then spin cells at 500 xg for 5 min.
d. Resuspend cell pellet in 3mLof R10 and count cells.
e. Rest cells at least 3 hours (up to overnight) at 2x106cells/mL in R10 medium + 1 µL/mL DNAse I at 37°C , 5% CO².
Note: during resting, prepare antibody cocktail master mix. Adjust volume of ECS for 50µL per test with FACS buffer.
f. After resting, add PBS up to 15mL or 50mL (whichever is closer, rounding up) to cells and transfer to 15mL or 50mL conical tube.
g. Spin cells at 500 xg for 5 minutes at room temperature (RT).
h. Resuspend cells in PBS to 1x107 cells/mL and count. If cells are too dilute, re-spin cells and resuspend at 1x107 cells/mL. Transfer 200mL of cells (2x106 cells) into each well of a V-bottom 96 well plate.
Viability and extracellular staining (ECS)
a. Spin plate at 500 xg for 5 minutes at RT
b. Using a multichannel pipette, carefully remove the supernatant.
c. Add 5µLof 1:60 Aqua viability dye directly to cell pellet and resuspend cells.
d . Incubate for 10 minutes at RT in the dark.
e. Add 50µLof ECS antibody cocktail to cells and incubate for 20 minutes at RT in the dark (prepare 1% PFA fixation buffer in the meantime).
f. Add 100µL of FACS buffer to each well and spin plate at 500 xg for 5 minutes. Remove supernatant.
Fixation, Permeabilization, and ICS Staining
a. Fix cells with 100µL of 1xFixation/permeabilization buffer (1 part concentrate + 3 parts diluent) for 30 minutes at RT in the dark.
Note : Make ICS abs here
b. Add 100µLof 1x perm/wash buffer and centrifuge 800 xg for 5 min.
c. Remove supernatant and add 100µLICS cocktail (made in perm/wash buffer, made with diH²O) to the cells.
d. Incubate for 1h at RT in the dark.
e. Add 100µL perm/wash buffer. Centrifuge at 800 xg for 5 min.
f. Discard supernatant and resuspend pellet in 200µL1% PFA and transfer to FACS tubes.
g. Wrap in aluminum foil and store at 4°C until flow cytometry.
