Shreffler:PBMC Isolation

Equipment and Supplies

• Beckman Coulter (Allegra™ X-12R) centrifuge or comparable vendor
• Hemacytometer (Reichert Bright line). Fisher # 02-671-5
• Nalgene Cryo Freezing Container filled with 2-propanol. Fisher #15350-50
• – 80° C Freezer
• Biological safety cabinet. Forma Scientific Model 1286 or comparable vendor

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• Blood collection and basic lab supplies. Gloves, tube racks, sharps containers, microscope, Kimwipes, ethanol squirt bottle, cell counter, calculator, pen, paper, beaker for decanting, pipet aid, balance, boxes to hold cryovials, timer
• Sterile 50 mL conical tubes. Fisher #14-432-22
• Sterile 15 mL conical tubes. Fisher #14-959-53A
• Ficoll Paque Plus [density 1.077 g/mL, endotoxin tested < 0.12 EU/mL] (Amersham Biosciences; StemCell Technologies)
• 50 mL Accuspin tubes, radiation sterilized. Sigma #A-2055
• 12 mL Accuspin tubes, radiation sterilized. Sigma #A-1805
• Sterile, graduated transfer pipets. Fisher # 13-711-20
• 1.8 mL sterile cryogenic vials. Fisher # 12-565-171N
• 5 mL snap top tube (BD Falcon 12 x 75 mm) for RAST sample. Fisher #14-959-11A (Falcon # 352063)
• Sterile Dulbecco’s Phosphate buffered saline (PBS), Ca++ and Mg++ free. VWR#45000-436
• RPMI – 1640 plus antibiotics (Expires 1 month after preparing)
• AIM V Media (w/HAS). Invitrogen # 12055-091 (Expires 1 month after opening)
• Freezing Medium A. Tissue culture tested, heat inactivated, filtered, Human AB serum – supplied by Univ WI
• Freezing Medium B. 20% DMSO in Freezing Medium A – Supplied by Univ WI
• Sterile 100 ul tips. Fisher # 21-381-8a.
• Non-sterile tips. Fisher # 21-197-8g
• Sterile Pasteur pipet (9”)
• Pipet, 10-100 ul. Fisher # 05-402-88
• Turk’s solution. VWR Catalog: #RC885016 – 500mL, #RC885032 – 1L

Isolation of Mononuclear Cells

(Sterile Technique)

This is a sterile procedure and all steps should be performed in a hood.

1. Turn on the hood. Bring Ficoll and PBS to 20°C in the hood.
2. Obtain whole blood specimens collected in sodium heparin (green top) collection tubes and record subject information, i.e. ID #, date collected, date received.
3. If performing Basophil Activation assay on this sample, set aside 3mL of whole blood.
4. Dilute remaining blood at 1:1 with PBS in 50 mL conical tubes.
5. Place 15 mL of Ficoll in a 50 mL conical tube. Overlay with up to 30mL of diluted blood, adding it very slowly to make sure that the blood doesn’t mix with the Ficoll layer.
6. Centrifuge at 500 g for 30 minutes at room temperature (slow acceleration, deceleration off to ensure no disruption of the density gradient).
7. Using a sterile transfer pipette, aspirate the buffy coat (peripheral blood mononuclear cells [PBMCs]) into a new 50 mL conical tube. (Avoid aspirating the Ficoll.) Add PBS to bring to a minimum of 2x the volume, inverting up and down to mix.
8. Centrifuge at 500 g for 20 minutes at room temperature (maximum acceleration and deceleration).
9. Aspirate and discard the supernatant. Resuspend the cell pellet first by tapping the tube until no clumps are visible, then adding 1 mL of PBS. Set aside a 10 µL aliquot of cells for counting as follows: place the 10 µL of cells into 90 µL of PBS in a small sterile eppendorf tube, and add 100 µL of Trypan Blue (0.2%) into the eppendorf tube. Mix well, and let it sit for 1 minute before counting.
10. Add 19 mL of PBS to the cells in 990 µL to make a total volume of ~20 mL and centrifuge at 300 g for 15 minutes at room temperature (maximum acceleration and deceleration).
11. In order to determine the volume to use for resuspending the PBMCs after the wash, the total number of cells in the sample must be determined.
12. Carefully introduce 10 µL of the stained cells into the notch of a hemocytometer and record cell counts using a hand-held counter. Calculate the number of cells, taking into account the dilution factors and sample volume used. Or place 20 µL of the stained cells onto a disposable slide and count using the automated cell counter.
13. After centrifugation is completed, aspirate and discard the supernatant. Resuspend the cell pellet by tapping the tube until no clumps are visible. Suspend PBMCs with PBS at 10 x 10⁶/ml in 15 ml PP conical tube.

• Microcentrifuge tubes. Fisher # 02-681-290
• Serological pipet, 25 mL. Fisher # 13-678-14b