TITLE: Spectral Flow Phenotyping of CD226 KO CD127- Tregs During Expansion
AUTHORS: Matthew Brown, Brusko Laboratory

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This Standard Operating Procedure provides instructions for staining surface and intracellular markers on primary cells to be used for spectral flow cytometry analysis. This SOP was designed for the purpose of staining isolated primary CD4</span><span style = "vertical-align:super;">+</span><span> CD25</span><span style = "vertical-align:super;">+</span><span> Tregs and Tconv cells for experiments pertaining to CRISPR KOs of CD226 on CD127</span><span style = "vertical-align:super;">-</span><span> Tregs. Cells will be stained for CD4, CD25, CD45RA, CD127, CD197, CD226, FoxP3, Helios, L/D NIR, & TIGIT.</span></div></div>

[STEPS]
?. [Preparing Falcon Tubes and Cells for Staining]
Transfer 100,000 Unelectroporated CD127- Tregs into the "CD127- Tregs" tube.
?. [Preparing Falcon Tubes and Cells for Staining]
Obtain the appropriate number of 5 mL Falcon Tubes  based on the cell subsets and timepoints being analyzed.
?. [Preparing Falcon Tubes and Cells for Staining]
Transfer 100,000 Unelectroporated CD127- Tregs into the "Unstained Control" tube.
?. [Preparing Falcon Tubes and Cells for Staining]
Transfer 100,000 Unelectroporated CD127- Tregs into the "Unstained Control" tube.
?. [Preparing Falcon Tubes and Cells for Staining]
Transfer 100,000 Electroporated CD127- Tregs into the "No Guide CD127- Tregs" tube.
?. [Preparing Falcon Tubes and Cells for Staining]
Transfer 100,000 Unelectroporated CD127- Tregs into the "Unelectroporated CD127- Tregs" tube.
?. [Preparing Falcon Tubes and Cells for Staining]
Transfer 100,000 CD226 KO CD127- Tregs into the "CD226 KO CD127- Tregs" tube.
?. [Preparing Falcon Tubes and Cells for Staining]
Transfer 100,000 CD127- Tregs into the "Unstained Control" tube.
?. [Preparing Falcon Tubes and Cells for Staining]
Transfer 100,000 CD127- Tregs into the "CD127- Tregs" tube.
?. [Preparing Falcon Tubes and Cells for Staining]
Transfer 100,000 CD226- Tregs into the "CD226- Tregs" tube.
?. [Conducting Live/Dead NIR Staining of Tregs]
Prepare  of   Live/Dead Near IR Working Solution by diluting  of reconstituted   to  of
1 mL
0.1 µl
1 mL
To reconstitute  , add   of DMSO provided within the kit to a single unopened tube of lyophilized dye.
?. [Conducting Live/Dead NIR Staining of Tregs]
Add   of  Live/Dead Near IR Working Solution to the "CD127- Tregs" tube.
1 mL
?. [Conducting Live/Dead NIR Staining of Tregs]
Add  of  to the "Unstained Control" tube.
1 mL
?. [Preparing Falcon Tubes and Cells for Staining]
Centrifuge all tubes for 7 minutes at 350 g .
Centrifuge: 350 34, 7 min, 23 10
?. [Preparing Falcon Tubes and Cells for Staining]
After centrifugation, decant the supernatant and proceed to viability staining.
?. [Preparing Falcon Tubes and Cells for Staining]
Centrifuge all tubes for 7 minutes at 350 g .
Centrifuge: 350 34, 7 min, 23 10
?. [Preparing Falcon Tubes and Cells for Staining]
After centrifugation, decant the supernatant and proceed to viability staining.
?. [Preparing Falcon Tubes and Cells for Staining]
Centrifuge all tubes for 7 minutes at 350 g .
Centrifuge: 350 34, 7 min, 23 10
?. [Preparing Falcon Tubes and Cells for Staining]
After centrifugation, decant the supernatant and proceed to viability staining.
?. [Conducting Live/Dead NIR Staining of Tregs]
Vortex all tubes for  to ensure complete resuspension of the cells within the dye (or PBS for the unstained control).
?. [Conducting Live/Dead NIR Staining of Tregs]
After vortexing, incubate all cells for  at   in the dark.
4 °C
?. [Conducting Live/Dead NIR Staining of Tregs]
After vortexing, centrifuge all tubes for 5 minutes at 450 g .
Centrifuge: 450 34, 5 min, 23 10
?. [Conducting Live/Dead NIR Staining of Tregs]
After centrifugation, decant the supernatant and proceed to extracellular staining.
?. [Conducting Live/Dead NIR Staining of Tregs]
Prepare  of   Live/Dead Near IR Working Solution by diluting  of reconstituted   to  of
3 mL
0.3 µl
3 mL
To reconstitute  , add   of DMSO provided within the kit to a single unopened tube of lyophilized dye.
?. [Conducting Live/Dead NIR Staining of Tregs]
Add   of  Live/Dead Near IR Working Solution to the "Unelectroporated CD127- Tregs" tube.
1 mL
?. [Conducting Live/Dead NIR Staining of Tregs]
Add   of  Live/Dead Near IR Working Solution to the "No Guide CD127- Tregs" tube.
1 mL
?. [Conducting Live/Dead NIR Staining of Tregs]
Add   of  Live/Dead Near IR Working Solution to the "CD226 KO CD127- Tregs" tube.
1 mL
?. [Conducting Live/Dead NIR Staining of Tregs]
Add  of  to the "Unstained Control" tube.
1 mL
?. [Conducting Live/Dead NIR Staining of Tregs]
Prepare  of   Live/Dead Near IR Working Solution by diluting  of reconstituted   to  of
2 mL
0.2 µl
2 mL
To reconstitute  , add   of DMSO provided within the kit to a single unopened tube of lyophilized dye.
?. [Conducting Live/Dead NIR Staining of Tregs]
Add   of  Live/Dead Near IR Working Solution to the "CD127- Tregs" tube.
1 mL
?. [Conducting Live/Dead NIR Staining of Tregs]
Add   of  Live/Dead Near IR Working Solution to the "CD226- Tregs" tube.
1 mL
?. [Conducting Live/Dead NIR Staining of Tregs]
Add  of  to the "Unstained Control" tube.
1 mL
?. [Conducting Live/Dead NIR Staining of Tregs]
Vortex all tubes for  to ensure complete resuspension of the cells within the dye (or PBS for the unstained control).
?. [Conducting Live/Dead NIR Staining of Tregs]
Vortex all tubes for  to ensure complete resuspension of the cells within the dye (or PBS for the unstained control).
?. [Conducting Live/Dead NIR Staining of Tregs]
After vortexing, incubate all cells for  at  .
4 °C
?. [Conducting Live/Dead NIR Staining of Tregs]
After vortexing, incubate all cells for  at  .
4 °C
?. [Conducting Live/Dead NIR Staining of Tregs]
After incubation, centrifuge all tubes for 5 minutes at 450 g .
Centrifuge: 450 34, 5 min, 23 10
?. [Conducting Live/Dead NIR Staining of Tregs]
After incubation, centrifuge all tubes for 5 minutes at 450 g .
Centrifuge: 450 34, 5 min, 23 10
?. [Conducting Live/Dead NIR Staining of Tregs]
After centrifugation, decant the supernatant and proceed to extracellular staining.
?. [Conducting Live/Dead NIR Staining of Tregs]
After centrifugation, decant the supernatant and proceed to extracellular staining.
?. [Conducting Extracellular Staining of Tregs]
Generate the Extracellular Phenotyping Cocktail as described below in a  :
?. [Conducting Extracellular Staining of Tregs]
Add  of
1 µl
?. [Conducting Extracellular Staining of Tregs]
Add  of
1 µl
?. [Conducting Extracellular Staining of Tregs]
Add  of
1 µl
?. [Conducting Extracellular Staining of Tregs]
Add  of
1 µl
?. [Conducting Extracellular Staining of Tregs]
Add  of
1 µl
?. [Conducting Extracellular Staining of Tregs]
Add  of
1 µl
?. [Conducting Extracellular Staining of Tregs]
Add  of
1 µl
?. [Conducting Extracellular Staining of Tregs]
After generating the Extracellular Phenotyping Cocktail, add all  to the "CD127- Tregs" tube.
7 µl
The "Unstained Control" tube should NOT recieve any of the antibody cocktail. Optionally,   of   may be added to the "Unstained Control" tube, but this is not necessary
?. [Conducting Extracellular Staining of Tregs]
Vortex all tubes for  to ensure complete resuspension of the cells within the antibody cocktail (or, optionally, PBS for the unstained control).
?. [Conducting Live/Dead NIR Staining of Tregs]
After incubation, add   of  to each tube.
1 mL
?. [Conducting Live/Dead NIR Staining of Tregs]
Vortex all tubes for  to ensure complete dilution of the dye (or PBS for the unstained control).
?. [Conducting Extracellular Staining of Tregs]
After vortexing, incubate all cells for  at   in the dark.
4 °C
?. [Conducting Extracellular Staining of Tregs]
After incubation, add   of  to each tube.
1 mL
?. [Conducting Extracellular Staining of Tregs]
Vortex all tubes for  to ensure complete dilution of the antibody cocktail (or, optionally, PBS for the unstained control).
?. [Conducting Extracellular Staining of Tregs]
After vortexing, centrifuge all tubes for 5 minutes at 450 g .
Centrifuge: 450 34, 5 min, 23 10
?. [Conducting Extracellular Staining of Tregs]
After centrifugation, decant the supernatant and proceed to fixation/permeabilization.
?. [Conducting Fixation/Permeabilization of Tregs]
Generate   1x Fixation/Permeabilization Buffer by combining  of 4x Fixation/Permeabilization Concentrate and  of Fixation/Permeabilization Diluent provided within the  .
400 µl
100 µl
300 µl
The 1x Fixation/Permeabilization Buffer should be stored at   when not actively in use.
?. [Conducting Extracellular Staining of Tregs]
Generate the Extracellular Phenotyping Cocktail as described below in a  :
?. [Conducting Extracellular Staining of Tregs]
Add  of
3 µl
?. [Conducting Extracellular Staining of Tregs]
Add  of
3 µl
?. [Conducting Extracellular Staining of Tregs]
Add  of
3 µl
?. [Conducting Extracellular Staining of Tregs]
Add  of
3 µl
?. [Conducting Extracellular Staining of Tregs]
Add  of
3 µl
?. [Conducting Extracellular Staining of Tregs]
Add  of
3 µl
?. [Conducting Extracellular Staining of Tregs]
Add  of
3 µl
?. [Conducting Extracellular Staining of Tregs]
After generating the Extracellular Phenotyping Cocktail, add the cocktail to each of the following tubes:
The "Unstained Control" tube should NOT recieve any of the antibody cocktail. Optionally,   of   may be added to the "Unstained Control" tube, but this is not necessary
?. [Conducting Extracellular Staining of Tregs]
Add  of the Extracellular Phenotyping Cocktail to the "Unelectroporated CD127- Tregs" tube.
7 µl
?. [Conducting Extracellular Staining of Tregs]
Vortex all tubes for  to ensure complete resuspension of the cells within the antibody cocktail (or, optionally, PBS for the unstained control).
?. [Conducting Extracellular Staining of Tregs]
After vortexing, incubate all cells for  at   in the dark.
4 °C
?. [Conducting Extracellular Staining of Tregs]
Add  of the Extracellular Phenotyping Cocktail to the "No Guide CD127- Tregs" tube.
7 µl
?. [Conducting Extracellular Staining of Tregs]
Add  of the Extracellular Phenotyping Cocktail to the "CD226 KO CD127- Tregs" tube.
7 µl
?. [Conducting Extracellular Staining of Tregs]
After incubation, vortex all cells for .
?. [Conducting Extracellular Staining of Tregs]
After vortexing, incubate all cells for  at   in the dark.
23 Room temperature
?. [Conducting Extracellular Staining of Tregs]
After incubation, vortex all cells for .
?. [Conducting Extracellular Staining of Tregs]
After vortexing, incubate all cells for  at   in the dark.
4 Room temperature
?. [Conducting Extracellular Staining of Tregs]
After incubation, add   of  to each tube.
1 mL
?. [Conducting Extracellular Staining of Tregs]
Vortex all tubes for  to ensure complete dilution of the antibody cocktail (or, optionally, PBS for the unstained control).
?. [Conducting Extracellular Staining of Tregs]
After vortexing, centrifuge all tubes for 5 minutes at 450 g .
Centrifuge: 450 34, 5 min, 23 10
?. [Conducting Extracellular Staining of Tregs]
After centrifugation, decant the supernatant and proceed to fixation/permeabilization.
?. [Conducting Fixation/Permeabilization of Tregs]
Add  of 1x Fixation/Permeabilization Buffer to all tubes.
100 µl
?. [Conducting Fixation/Permeabilization of Tregs]
Vortex all tubes for  to ensure complete resuspension of the cells within the 1x Fixation/Permeabilization Buffer.
?. [Conducting Fixation/Permeabilization of Tregs]
After vortexing, incubate all cells for  at  in the dark.
0 Room temperature
?. [Conducting Fixation/Permeabilization of Tregs]
After incubation, add  of  to all tubes.
1 mL
?. [Conducting Fixation/Permeabilization of Tregs]
Vortex all tubes for  to ensure complete dilution of the 1x Fixation/Permeabilization Buffer.
?. [Conducting Fixation/Permeabilization of Tregs]
After vortexing, centrifuge all tubes for 5 minutes at 450 g .
Centrifuge: 450 34, 5 min, 23 10
?. [Conducting Fixation/Permeabilization of Tregs]
Conduct a second 1x Fixation/Permeabilization Buffer wash.
?. [Conducting Fixation/Permeabilization of Tregs]
Generate   1x Permeabilization Buffer by combining  of 10x Permeabilization Concentrate provided within the  and  of   .
2 mL
0.2 mL
1.8 mL
The 1x Permeabilization Buffer should be stored at   when not actively in use.
?. [Conducting Fixation/Permeabilization of Tregs]
Add  of 1x Fixation/Permeabilization Buffer to all tubes.
1 mL
?. [Conducting Fixation/Permeabilization of Tregs]
Vortex all tubes for  to ensure complete resuspension of the cells within the 1x Permeabilization Buffer.
?. [Conducting Fixation/Permeabilization of Tregs]
After vortexing, incubate all cells for  at  in the dark.
0 Room temperature
?. [Conducting Fixation/Permeabilization of Tregs]
After incubation, add  of  to all tubes.
1 mL
?. [Conducting Fixation/Permeabilization of Tregs]
Vortex all tubes for  to ensure complete dilution of the 1x Permeabilization Buffer.
?. [Conducting Fixation/Permeabilization of Tregs]
After vortexing, centrifuge all tubes for 5 minutes at 450 g .
Centrifuge: 450 34, 5 min, 23 10
?. [Conducting Fixation/Permeabilization of Tregs]
After centrifugation, decant the supernatant from all tubes and proceed to intracellular staining.
?. [Conducting Fixation/Permeabilization of Tregs]
After centrifugation, decant the supernatant from all tubes.
?. [Conducting Fixation/Permeabilization of Tregs]
Generate   1x Fixation/Permeabilization Buffer by combining  of 4x Fixation/Permeabilization Concentrate and  of Fixation/Permeabilization Diluent provided within the  .
800 µl
200 µl
600 µl
The 1x Fixation/Permeabilization Buffer should be stored at   when not actively in use.
?. [Conducting Fixation/Permeabilization of Tregs]
Add  of 1x Fixation/Permeabilization Buffer to all tubes.
100 µl
?. [Conducting Fixation/Permeabilization of Tregs]
Vortex all tubes for  to ensure complete resuspension of the cells within the 1x Fixation/Permeabilization Buffer.
?. [Conducting Fixation/Permeabilization of Tregs]
After vortexing, incubate all cells for  at  in the dark.
0 Room temperature
?. [Conducting Fixation/Permeabilization of Tregs]
After incubation, add  of  to all tubes.
1 mL
?. [Conducting Fixation/Permeabilization of Tregs]
Vortex all tubes for  to ensure complete dilution of the 1x Fixation/Permeabilization Buffer.
?. [Conducting Fixation/Permeabilization of Tregs]
After vortexing, centrifuge all tubes for 5 minutes at 450 g .
Centrifuge: 450 34, 5 min, 23 10
?. [Conducting Fixation/Permeabilization of Tregs]
After centrifugation, decant the supernatant from all tubes.
?. [Conducting Fixation/Permeabilization of Tregs]
Conduct a second 1x Fixation/Permeabilization Buffer wash.
?. [Conducting Fixation/Permeabilization of Tregs]
Generate   1x Permeabilization Buffer by combining  of 10x Permeabilization Concentrate provided within the  and  of   .
2 mL
0.2 mL
1.8 mL
The 1x Permeabilization Buffer should be stored at   when not actively in use.
?. [Conducting Fixation/Permeabilization of Tregs]
Add  of 1x Fixation/Permeabilization Buffer to all tubes.
1 mL
?. [Conducting Fixation/Permeabilization of Tregs]
Vortex all tubes for  to ensure complete resuspension of the cells within the 1x Permeabilization Buffer.
?. [Conducting Fixation/Permeabilization of Tregs]
After vortexing, incubate all cells for  at  in the dark.
0 Room temperature
?. [Conducting Fixation/Permeabilization of Tregs]
After incubation, add  of  to all tubes.
1 mL
?. [Conducting Fixation/Permeabilization of Tregs]
Vortex all tubes for  to ensure complete dilution of the 1x Permeabilization Buffer.
?. [Conducting Fixation/Permeabilization of Tregs]
After vortexing, centrifuge all tubes for 5 minutes at 450 g .
Centrifuge: 450 34, 5 min, 23 10
?. [Conducting Fixation/Permeabilization of Tregs]
After centrifugation, decant the supernatant from all tubes and proceed to intracellular staining.
?. [Conducting Intracellular Staining of Tregs]
Generate the Intracellular Phenotyping Cocktail as described below in a  :
?. [Conducting Intracellular Staining of Tregs]
Add  of
1.25 µl
?. [Conducting Intracellular Staining of Tregs]
Add  of
1.25 µl
?. [Conducting Intracellular Staining of Tregs]
After generating the Intracellular Phenotyping Cocktail, add all  to the "CD127- Tregs" tube.
5 µl
The "Unstained Control" tube should NOT recieve any of the antibody cocktail. Optionally,   of   may be added to the "Unstained Control" tube, but this is not necessary
?. [Conducting Intracellular Staining of Tregs]
Vortex all tubes for  to ensure complete resuspension of the cells within the antibody cocktail (or, optionally, PBS for the unstained control).
?. [Conducting Intracellular Staining of Tregs]
After vortexing, incubate all cells for  at   in the dark.
4 °C
?. [Conducting Intracellular Staining of Tregs]
After incubation, vortex all cells for .
?. [Conducting Intracellular Staining of Tregs]
After vortexing, incubate all cells for  at   in the dark.
23 Room temperature
?. [Conducting Intracellular Staining of Tregs]
After incubation, add   of  to each tube.
1 mL
?. [Conducting Intracellular Staining of Tregs]
Vortex all tubes for  to ensure complete dilution of the antibody cocktail (or, optionally, PBS for the unstained control).
?. [Conducting Intracellular Staining of Tregs]
After vortexing, centrifuge all tubes for 5 minutes at 450 g .
Centrifuge: 450 34, 5 min, 23 10
?. [Conducting Intracellular Staining of Tregs]
After centrifugation, decant the supernatant and add  of to all tubes, then proceed to collect data using the .
50 µl
Cells may be stored at   until acquisition, however, acquisition must be completed within 48 hours of fixation to ensure sufficient data quality.
?. [Conducting Intracellular Staining of Tregs]
Generate the Intracellular Phenotyping Cocktail as described below in a  :
?. [Conducting Intracellular Staining of Tregs]
After generating the Intracellular Phenotyping Cocktail, add the cocktail to the following tubes.
The "Unstained Control" tube should NOT recieve any of the antibody cocktail. Optionally,   of   may be added to the "Unstained Control" tube, but this is not necessary
?. [Conducting Intracellular Staining of Tregs]
Vortex all tubes for  to ensure complete resuspension of the cells within the antibody cocktail (or, optionally, PBS for the unstained control).
?. [Conducting Intracellular Staining of Tregs]
After vortexing, incubate all cells for  at   in the dark.
4 °C
?. [Conducting Intracellular Staining of Tregs]
After incubation, vortex all cells for .
?. [Conducting Intracellular Staining of Tregs]
After vortexing, incubate all cells for  at   in the dark.
23 Room temperature
?. [Conducting Intracellular Staining of Tregs]
After incubation, add   of  to each tube.
1 mL
?. [Conducting Intracellular Staining of Tregs]
Vortex all tubes for  to ensure complete dilution of the antibody cocktail (or, optionally, PBS for the unstained control).
?. [Conducting Intracellular Staining of Tregs]
After vortexing, centrifuge all tubes for 5 minutes at 450 g .
Centrifuge: 450 34, 5 min, 23 10
?. [Conducting Intracellular Staining of Tregs]
After centrifugation, decant the supernatant and add  of to all tubes, then proceed to collect data using the .
50 µl
Cells may be stored at   until acquisition, however, acquisition must be completed within 48 hours of fixation to ensure sufficient data quality.
?. [Conducting Intracellular Staining of Tregs]
Add  of
2.5 µl
?. [Conducting Intracellular Staining of Tregs]
Add  of
3.75 µl
?. [Conducting Intracellular Staining of Tregs]
Add  of
3.75 µl
?. [Conducting Intracellular Staining of Tregs]
Add  of
7.5 µl
?. [Conducting Intracellular Staining of Tregs]
Add  of the Intracellular Phenotyping Cocktail to the "Unelectroporated CD127- Tregs" tube.
5 µl
?. [Conducting Intracellular Staining of Tregs]
Add  of the Intracellular Phenotyping Cocktail to the "No Guide CD127- Tregs" tube.
5 µl
?. [Conducting Intracellular Staining of Tregs]
Add  of the Intracellular Phenotyping Cocktail to the "CD226 KO CD127- Tregs" tube.
5 µl