Isolation of PBMC’s from whole blood:
Histopaque procedure: This procedure should be done in the cell culture hood to maintain
sterility.
1. To a 15-ml conical centrifuge tube, add 6.0 ml HISTOPAQUE®-1077 and bring to room
temperature.
2. Carefully layer 6.0 ml whole blood onto the HISTOPAQUE®-1077. Centrifuge at 400 x g for
exactly 30 minutes at room temperature. Centrifugation at lower temperatures, such as 4°C, may
result in cell
clumping and poor recovery.
3. After centrifugation, carefully aspirate, with a Pasteur pipet, the upper layer to within 0.5 cm
of the opaque interface containing mononuclear cells. Discard upper layer.
4. Carefully transfer the opaque interface, with a Pasteur pipet, into a clean conical centrifuge
tube.
5. Add to this tube (Step 4), 5 ml sterile Phosphate Buffered Saline (PBS) and mix by gentle
aspiration.
6. Centrifuge at 250 x g for 10 minutes.
7. Aspirate the supernatant and discard.
8. Resuspend cell pellet with 10% DMEM with P/S and add to cell culture plate.
Staining Cell Surface Antigens/Extracellular Flow Staining
Recipes:
Staining Buffer (50ml PBS, .045g Sodium azide (0.09%), 2.5ml FBS(5%))
CONTROLS NEEDED – 1) unstained controls and 2) single stained controls needed
1. Remove cells from each well (2 ml), either through deconting (suspension cells) or with
trypsin (adherent cells) and place in 15 ml eppendorf tube. Spin at 1500 rpm’s for 5 mins, decant
supernatant and resuspend the pellet in 100 ul of Staining Buffer. Pulse vortex gently to mix and
break up pellet.
2. If staining granulocytes (neutrophils, macrophages) add 20μL FcBlock in the 100μL of
staining buffer and incubate for 10min @ 4°C. If staining other cells proceed to step 3.
3. Remove 50 ul of cells (with or without Fc Block) and incubate in a 1.5 ml eppendorf tube.
Add appropriate amount of antibody into almost all tubes (one no antibody control needed, other
controls may be needed). Incubate for at least 30 minutes in the dark on ice or at 4°C.
4. Add 200 ul of Flow Cytometry Staining Buffer to each tube tostop the reaction. Pellet the cells
by centrifugation at 5000 rpm for 5 minutes. Decant the supernatant. Resuspend the pellet in 100
ul of staining buffer. Pulse vortex to resuspend the pellet. If also staining intracellular antigens
proceed to step 6.
5. Add 5 ul of Propidium iodide (1 mg/ml solution) to identify dead cells. Run on flow
cytometer.
Intracellular Staining
1. Fix the cells by adding 100 μL of IC Fixation Buffer (eBioscience) while vortexing the tube.
2. Incubate in the dark at room temperature for 20 minutes.
3. Without washing, add 1 mL of 1X Permeabilization Buffer (eBioscience) to each tube.
4. Centrifuge samples at 5000 rpm at room temperature for 5 minutes, then discard the
supernatant.
5. Resuspend the cell pellet in 1 mL of 1X Permeabilization Buffer. RESUPSEND THE
PELLET!
6. Centrifuge samples at 5000 rpm at room temperature for 5 minutes, then discard the
supernatant.
7. Resuspend the cells in 100μL of 1X Permeabilization Buffer to each tube. Add appropriate
amount of antibody per sample and incubate in the dark at room temperature for 20 minutes.
8. Add 1 mL of 1X Permeabilization Buffer to each tube.
9. Centrifuge samples at 5000 rpm at room temperature for 5minutes, then discard the
supernatant.
10. Add 1 mL of Flow Cytometry Staining Buffer to each tube.
11. Run on flow cytometer. 30,000 events per sample