Apr 20, 2024

Public workspaceFlow Cytometry ECS Surface Antigens V.2

DOI
dx.doi.org/10.17504/protocols.io.dm6gpwq11lzp/v2
Flow Cytometry ECS Surface Antigens
  • Michael Betts1,
  • Gregory Golden1
  • 1University of Pennsylvania
Sandy Beshir
Open access
DOI: dx.doi.org/10.17504/protocols.io.dm6gpwq11lzp/v2
Protocol Citation: Michael Betts, Gregory Golden 2024. Flow Cytometry ECS Surface Antigens. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpwq11lzp/v2Version created by Sandy Beshir
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 20, 2024
Last Modified: April 20, 2024
Protocol Integer ID: 98531
Keywords: extracellular epitope staining (ECS), intra-cellular epitope staining (ICS), HIRN
Funders Acknowledgement:
NIH
Grant ID: U01 DK112217
Abstract
High-parameter flow cytometry enables identification and characterization of a wide range of cell populations within a biological sample. Analysis of cell-surface epitopes using a collection of fluorophore-labeled antibodies, or extracellular stain (ECS), sufficiently labels population-identifying markers for further analysis with flow cytometry. Importantly, ECS 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 procedures for cell-surface epitope labeling.
Materials
Materials Required

1. Reagent1x phosphate saline buffer (PBS) Corning 21-031-CMCorningCatalog #21-031-CM

2. ReagentBovine serum albumin (BSA) Gemini 700-101PGemini BioproductsCatalog #700-101P

3. ReagentSodium AzideFisher ScientificCatalog #S2271-500

4. FACS buffer (1xPBS, 10g/L BSA, 1 g/L sodium azide)

5. ReagentRPMICorningCatalog #10-040-CM

6. ReagentFetal calf serum (FCS)Gemini BioproductsCatalog # 900-108

7. ReagentPenicillin/streptomycin 10000 U/mL penicillin 10000 µg/mL streptomycinLonzaCatalog #17-602E
Lonza (RRID:SCR_000377)

8. “R10” medium (RPMI, 10% FCS, 1% penicillin/streptomycin)

9. ReagentDNAse IRocheCatalog #04716728001

10. ReagentLive/Dead fixable Aqua Dead Cell Stain kitInvitrogen - Thermo FisherCatalog #L34966
diluted 1:60 in 1x PBS (prepare fresh each day from DMSO stock)

11. ReagentParaformaldehyde (PFA)Electron Microscopy SciencesCatalog #15712-S diluted to 1% in PBS

12. Fluorophore-labelled antibodies of choice

13. Hemocytometer



Procedure
Procedure
Thawing and Resting

a. Pre-warm R10 media in a Temperature37 °C water bath.

b. Thaw samples in-vial using a Temperature37 °C water bath.

c. Add thawed cells to Amount14 mL of R10, then spin cells at 500 xg for 5 min.

d. Resuspend cell pellet in Amount3 mL of R10 and count cells.

e. Rest cells at least 3 hours (up to overnight) at 2x106 cells/mL in R10 medium + 1 µL/mL DNAse I at Temperature37 °C , 5% CO².
Note: during resting, prepare antibody cocktail master mix. Adjust volume of ECS for Amount50 µL per test with FACS buffer.

f. After resting, add PBS up to Amount15 mL or Amount50 mL (whichever is closer, rounding up) to cells and transfer to Amount15 mL or Amount50 mL 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 Amount200 mL of cells (2x106cells) 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. Using a multichannel pipette, add Amount45 µL of PBS to well.

d. Add Amount5 µL of 1:60 Aqua viability dye directly to cell pellet and resuspend cells.

e. Incubate for 10 minutes at RT in the dark.

f. Add Amount50 µL of ECS antibody cocktail to cells and incubate for 20 minutes at RT in the dark (prepare 1% PFA fixation buffer in the meantime).

g. Add Amount100 µL of FACS buffer to each well and spin plate at 500 xg for 5 minutes.

h. Discard supernatant and fix cells with Amount200 µL of 1% PFA.

i. Run samples on flow cytometer.