TITLE: PBMC Isolation & Cryopreservation from Whole Blood
AUTHORS: cecilia , Alessandro Sette

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this procedure is to isolate and cryopreserve peripheral blood mononuclear cells (PBMC's) from 30 mL of freshly drawn whole blood.</div></div>

[STEPS]
?. [Step 1: Prepare materials and supplies]
Place RPMI media and Ficoll in water bath. Bring to  ( - ).
0 Room temperature
19 °C
25 °C
?. [Step 2: First Spin]
Spin tubes at  (breaks off; 5 acc 0 brake) for plasma removal.Total time for spin including acceleration and deceleration – .
Centrifuge: 1850 33, 15 min
?. [Step 3: Plasma Aliquot]
Pipette  from the spun blood. Leave ~ -  above the buffycoat/RBC layers.
[plasma]
1 mL
[plasma]
?. [Step 3: Plasma Aliquot]
Store sample ID labeled Plasma aliquot in the -80˚C freezer.
?. [Step 4: Preparing Ficoll tubes (15 mL of Ficoll to 35 mL of diluted blood) for density gradient centrifugation]
Prepare two sterile 50 mL conical tubes and label with sample ID.
?. [Step 4: Preparing Ficoll tubes (15 mL of Ficoll to 35 mL of diluted blood) for density gradient centrifugation]
Use pipette gun to distribute  into each tube.
[Ficoll]
?. [Note to step 5-9]
Use a maximum of 10-12 Ficoll tubes at one single time. If more samples – repeat step 5-9 with remainder while the first batch waits for step 10 (Fourth spin). Try to limit the time the cells are in contact with Ficoll.
?. [Step 5: Dilute Blood Ficoll overlay]
Aspirate remaining plasma with Pasteur pipette vacuum from all blood tubes until 1 mL - 2 mL of plasma remain above RBC layers.
?. [Step 5: Dilute Blood Ficoll overlay]
Dilute remaining blood in all tubes with RPMI media to 50 mL.
?. [Step 5: Dilute Blood Ficoll overlay]
Gently pipet diluted blood to homogenize.
Important to pipet slowly.
?. [Step 5: Dilute Blood Ficoll overlay]
Use pipette gun to slowly dispense  onto each Ficoll tube, without breaking the interface or mixing. ( to )
[diluted blood]
[diluted blood]
[Ficoll]
?. [Step 5: Dilute Blood Ficoll overlay]
Use small amount of RPMI media to rinse empty blood tubes and overlay over Ficoll.
?. [Step 5: Dilute Blood Ficoll overlay]
Secure all caps to avoid leaks.
?. [Step 6: Second spin]
Spin tubes at  (breaks off). Total time for spin including acceleration and deceleration – .
Centrifuge: 1850 33, 25 min
?. [Step 7: Harvesting PBMC buffy coat and washing cells]
Prepare and label new sterile conical tube for PBMC’s; 2 isolated layers per conical tube(i.e. 2 ficoll tubes / 2 layers = 1 50ml conical needed).
?. [Step 7: Harvesting PBMC buffy coat and washing cells]
Retrieve Ficoll tubes from centrifuge. Tubes will have four distinct layers.
?. [Step 7: Harvesting PBMC buffy coat and washing cells]
Aspirate media/plasma (top layer) from all tubes until 10 mL remain over PBMC buffy coat layer.
?. [Step 7: Harvesting PBMC buffy coat and washing cells]
Pipette PBMC buffy coat layers into sterile 50 mL conical tubes (combine 2 isolated layers per tube).
?. [Step 7: Harvesting PBMC buffy coat and washing cells]
Rinse conical tube containing PBMC’s with RPMI media by adding RPMI to 50 mL.
?. [Step 8: Third spin]
Spin tubes at  (breaks low).
Centrifuge: 1850 33, 10 min
?. [Step 9: Washing cells]
Aspirate supernatant and resuspend cell pellets in .
[RPMI media]
?. [Step 9: Washing cells]
Wash cells by diluting to 50 mL with RPMI media.
?. [Step 10: Fourth spin]
Spin tubes at  (breaks ON; acceleration at 5).
Centrifuge: 1850 33, 10 min
?. [Step 11: Washing cells and counting]
Aspirate supernatant and resuspend cell pellets in .
[RPMI media]
?. [Step 11: Washing cells and counting]
Combine cell pellets into one conical tube and dilute cell suspension to 30 mL.
?. [Step 11: Washing cells and counting]
Cap and invert tube to ensure cell suspension is homogenous.
?. [Step 11: Washing cells and counting]
Use p100 micropipette and aliquot  for counting.
[cell suspension]
?. [Step 11: Washing cells and counting]
Do a 1:2 dilution with  and count in hemocytometer (i.e.  with ).
[trypan blue]
[cell suspension]
[0.1% trypan blue]
?. [Step 12: Fifth spin]
Spin tubes at  (breaks OFF).
Centrifuge: 800 33, 10 min
?. [Step 13: Cryopreservation]
Print labels with sample ID, cell density, date, and initial. Prepare and label cryovials and Mr. Frosty container.
?. [Step 13: Cryopreservation]
Freeze at a density of ~10 million cells per vial with a maximum of 3 vials per sample for 30 ml sample.
?. [Step 13: Cryopreservation]
Aspirate supernatant.
?. [Step 13: Cryopreservation]
Resuspend cell pellet in appropriate amount of fridge cold Freezing media (FBS with ) at  per vial (i.e. 3 vials = resuspend in ).
[DMSO]
1 mL
[FBS]
Important to work quickly and in batches for this step. DMSO is toxic for the cells.
?. [Step 9: Washing cells]
Repeat step 5-9 as needed depending on number of samples being processed simultaneously.
?. [Step 11: Washing cells and counting]
Count the viable cells in 4 corner squares and middle square of hemocytometer.
?. [Step 11: Washing cells and counting]
Count the number of dead (blue) cells in the same squares.
?. [Step 11: Washing cells and counting]
Calculate cell density, total cells yielded and viability (no. of dead cells divided by total number of cells).
?. [Step 11: Washing cells and counting]
Calculate number of cryovials needed based on specified freezing density.
?. [Step 13: Cryopreservation]
** pipette cell suspension into cryovials quickly and efficiently to prevent loss of viability.
?. [Step 13: Cryopreservation]
Aim for 8-10 million cells per vial. - 15 million or less cells – 1 vial, - 16-23 million cells – 2 vials, - 24 million and above – 3 vials.
?. [Step 13: Cryopreservation]
Place cryovials in frosty container and freeze in -80˚C freezer ().
?. [Step 13: Cryopreservation]
Transfer cells to nitrogen tank for long term storage (work quickly to avoid temperature changes).