Flow cytometry of PBMCs

Mouse Bleed in the Animal Facility 

Using heparinized capillary tubes, scruff the mouse and perform retro-orbital bleeds 

Place ~200uL (4x full capillary tube) of blood into the 1.5mL tubes with heparin and gently mix 

Prepare Cells 

*PERFORM THE FOLLOWING STEPS ONE SAMPLE AT A TIME* 

Add 100uL of mouse blood into 15mL tubes containing 9mL H₂O *Don’t need to rinse pipette 

Quickly cap and invert tube 3 times 

After ~10 sec, add 1mL of 10X PBS to the tube and invert tube 3 times 

Spin samples for 10 min at 1500 rpm 

Aspirate the supernatant and keep pellet at the bottom 

If necessary, aspirate supernatant until 100-200uL remain. Remove the remaining supernatant using a pipette to prevent aspiration of the pellet 

Add 200uL of flow buffer to each 15mL tube 

Spin remaining mouse blood (in the 1.5mL tube with heparin) for 5 min at 3000 g 

Transfer plasma to a 0.6mL tube and store in the -80°C for future use 

Transfer 

Following the 96-well plate sample layout sheet, transfer the 200uL samples in the 15mL tubes to a 96 well plate 

Spin plates for 3 min at 1200 rpm 

Remove supernatant (flick out into sink), leaving plate facing down, blot on paper towel to remove residual flow buffer 

Prepare Diluted Fc Block (according to the excel spreadsheet calculations) and add 75uL/well and mix 

Incubate plate at 4°C for 15 min 

Prepare Extracellular staining (according to the empirically derived dilutions) and add 25uL/well and mix 

Incubate at 4°C for 15 min 

Wash with 100uL/well of flow buffer 

Spin plate for 3 min at 1200 rpm 

Remove supernatant (flick out into sink), leaving plate facing down, blot on paper towel to remove residual flow buffer 

Wash with 200uL/well of flow buffer 

Spin plate for 3 min at 1200 rpm 

Remove supernatant (flick out into sink), leaving plate facing down, blot on paper towel to remove residual flow buffer 

IF no intracellular staining: 

Same Day Flow -> continue to Final Washes and Transfer 

Next Day Flow -> add 1% formalin for overnight fixation (180uL FB + 20uL 10% Formalin/sample) 

IF panel has intracellular staining: 

Prepare Fix/Perm Buffer and add 200uL/well 

1:3 dilution (small brown: larger clear bottles) 

Incubate plate at 4°C for 30 min or OVERNIGHT INCUBATION 

Prepare Intracellular Stains 

Spin plate for 5 min at 400 g 

Remove supernatant (flick out into sink), leaving plate facing down, blot on paper towel to remove residual flow buffer 

Prepare permeabilization Buffer (according to the excel spreadsheet calculations) and add 200uL/well 

1:9 dilution (10x perm buffer: water)  

Prepare Intracellular Staining (according to the empirically derived dilutions) and add 100uL/well 

Prepare the stains using 1x permeabilization buffer  

Incubate plate at 4°C for 30 min  

Final Washes and Transfer 

Prepare flow tubes by adding 200uL of flow buffer into each tube 

Add 100uL/well of perm buffer to the plate and spin plate for 3 min at 1200 rpm 

Remove supernatant (flick out into sink), leaving plate facing down, blot on paper towel to remove residual flow buffer 

Add 200uL/well of perm buffer to the plate and spin plate for 3 min at 1200 rpm 

Remove supernatant (flick out into sink), leaving plate facing down, blot on paper towel to remove residual flow buffer 

Re-suspend samples with 200uL of flow buffer/sample in the plate and transfer to flow tubes 

Flow tubes should have a final volume of 400uL