Isolation of peripheral blood mononuclear cells (PBMCs) from whole blood and thawing

Jagannathan Lab, Stanford University

Savannah Nicole Lewis (snlewis@stanford.edu)

I: Isolating PBMCs from whole blood

-Materials:

• Ficoll (room temperature)
• 1X PBS
• R10 media:
  • 500mL RPMI-1640, 5mL penicillin/streptomycin, 5mL L-glutamine, 5mL HEPES, 50mL fetal bovine serum

-Protocol:

• Transfer 15mL blood to a 50mL conical tube
• Add equal volume of PBS to 50mL conical and mix thoroughly
• Add 10mL Ficoll to a clean, separate 50mL conical tube
• Slowly and gently later the blood/PBS over the Ficoll: touch serological pipette tip to the wall of the tube and allow blood to slowly run down the side of it
• Centrifuge at 2000rpm for 30 minutes at room temperature: slow start, no breaks
• Harvest PBMC layer using a 10mL serological pipette and move to a new 50mL conical tube
  • Layers separate as follows (top to bottom): serum, PBMCs, Ficoll, RBCs and granulocytes
  • Okay to remove PBS or Ficoll, but avoid taking RBCs (if you do/pelleted cells are red, add ACK lysis buffer, incubate for 5 minutes, then wash)
• Fill 50mL conical tube to the top with PBS, then centrifuge (1400rpm, 10 minutes)
• Aspirate supernatant, leaving 1-2mL to avoid disturbing PBMC pellet
• Gently resuspend pelleted cells with 1mL PBS, then fill 50mL conical to top with PBS
• Centrifuge (1400rpm, 10 minutes)
• (Repeat aspiration, washing, and centrifugation for a total of three washes)
• After the third wash, cells are ready to be frozen (any concentration) or rested in plates/flasks (~1M/mL)

II: Thawing PBMCs

-Materials:

• R10 media: pre-warmed (48°C)
• DNase
• Trypan Blue dye

-Protocol:

• Set water bath to 37°C
• Pre-label 15mL conical tubes with corresponding sample IDs
• Aliquot 5mL warmed R10 into labeled 15mL conical tubes:
  • Few samples: add 15uL DNase directly to PBMC cryovials
  • Many samples: add 15uL DNase into conical tubes
• Keep PBMCs on dry ice until ready to thaw
• When ready, load into a tube holder cassette and swish gently through warmed water bath until a pea-sized ice chunk remains
• Using P1000 pipette set to 1mL, gently mix PBMCs, then add slowly and dropwise to 15mL conical
• Rinse cryovial with 1mL R10, then add slowly and dropwise to 15mL conical
• Raise total 15mL conical volume to 10mL in R10 then centrifuge (1500rpm, 5 minutes)
• Aspirate most of the supernatant, leaving ~200uL left
• Gently resuspend pelleted PBMCs with 1mL R10
• Raise total 15mL conical volume to 10mL in R10 then centrifuge (1500rpm, 5 minutes)
• Aspirate as much supernatant as possible off the pelleted PBMCs
• Gently resuspend pelleted PBMCs with 1mL R10
• Remove 10uL resuspended PBMCs, add to 10uL Trypan Blue, and count the number of cells in each sample either manually (hemocytometer) or automatically (Countess Cell Counter)
• Raise total 15mL conical volume to 10mL in R10 and centrifuge (1500rpm, 5 minutes)
• Remove as much supernatant as possible, resuspend according to counting calculations
• Plate resuspended PBMCs according to experimental design in 96-well plate at a total per well volume of 200uL
• Place in incubator (37°C, 5% CO₂ ) and rest up to overnight but minimum one hour