Freezing Protocol for Human PBMC - Cellular Technology, Ltd

Freezing Protocol for Human PBMC
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Following is the recommended Protocol for freezing human PBMC in CTL-Cryo ABC
Serum-free Freezing Medium. This protocol provides all the necessary procedures to
successfully freeze and thaw human PBMC for subsequent use in biological assays.
Isolation of PBMC with Ficoll Density Gradient Centrifugation
• Draw venous blood using sodium heparin as an anti-coagulant (e.g., using green vacutainer tubes). Mix gently but
thoroughly by inverting the tubes. Process blood promptly. Pool the heparinized blood of each donor and dilute blood
at a 1:1 ratio using Ca2+ / Mg2+ free PBS (e.g., add 10 ml PBS to 10 ml blood). Use 50ml conical tubes or tissue
culture flasks for the dilution. Gently mix the blood with the PBS by inverting the tubes/flasks three times. Prepare
separate 50ml conical tubes. Label each tube and its corresponding lid with a unique identifier. Pipette 15ml of Ficoll
into each tube. Gently overlay the Ficoll with 25-30ml of the diluted blood using a sterile serological pipette. Avoid
mixing of the two phases. (Alternately, the blood can be added first and the Ficoll underlain with a serological pipette.)
• Carefully balance the weight of the tubes before centrifuging.
• Centrifuge the samples at 740g for 30 minutes. Make sure that the brake is off so that the deceleration does not
disrupt the density gradient.
• Accelerate the centrifuge slowly so that the gradients do not mix. If the centrifuge starts shaking, stop immediately but
gently; reweigh and balance the tubes.
• After the Ficoll centrifugation run is complete, immediately after the centrifuge stops, collect the mononuclear cells
from the plasma/Ficoll interface with a serological pipette. Transfer the cells from each Ficoll tube into a different sterile
50ml conical tube. Do not pool cells yet. While collecting the cells, be sure to aspirate plasma only and as little Ficoll as
possible. (If the amount of Ficoll in the wash tube is >5 ml, significant cell loss will occur). Fill each tube to the 30ml
mark with warm Ca2+ -free PBS. Do not fill to the 50ml mark as this will cause a lower cell yield.
• Centrifuge cells, this time at 330g for 10 minutes, at room temperature. This time the brake of the centrifuge should be
on, and acceleration should be high.
• As soon as centrifugation is complete, decant and discard the supernatant.
• Resuspend the cell pellet by tapping the tube until no clumps are visible. Promptely add CTL-Wash Medium (properly
diluted with RPMI-1640), using 5ml per 50ml tube. Pool cells of the same donor.
• Count the cells under a UV microscope with Acridine Orange (AO)/Ethidium Bromide (EB) for the most accurate
results (counting with Trypan Blue frequently causes overestimation by including apoptotic cells and erythrocytes).
The expected cell count for healthy human donors is 1-2 million PBMC for each ml of blood drawn.
• Centrifuge cells one more time at 330g for 10 minutes at room temperature.
• Decant supernatant, resuspend the cell pellet, and take up cells in the final media while adjusting for the desired
cell density:
† If a bioassay, e.g., and ELISPOT assay is the intended next step, use CTL-Test Medium that has been supplemented
with 1% fresh L-glutamine less than 7 days before. Adjust the cell concentration, e.g., to 3million/ml if 300,000
PBMC are to be plated per well in 100μl.
† If cryopreservation is the intended next step, use CTL-CryoABC. Adjust the cell concentration to 20million/ml.
Cellular Technology Limited • 20521 Chagrin Boulevard • Shaker Heights, OH 44122-5350 USA
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Freezing Protocol for Human PBMC
Cryopreservation of PBMC
In preparation of freezing (it is advisable to start with these preparations while the Ficoll gradient runs):
• Mix CTL-Cryo A with CTL-Cryo B in an 80%-20% (v/v) ratio (4+1) by slowly adding CTL-Cryo B into CTL-Cryo A
(CTL-Cryo B contains DMSO as a component, please refer to MSDS at the end of protocol). Sterile-filter the solution
(0.2µm filter).
• Label 1.8ml cryotubes. The number of tubes can be estimated from the anticipated cell yield (1-2million PBMC per ml
blood drawn) when freezing 10million PBMC per vial. Freezing more cells per vial may lead to cell loss.
After Washing of PBMC:
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• The PBMC have been resuspended in CTL- Cryo C medium at 20 million cells per ml (see above).
• Slowly, over a time period of ~2 min, add an equal volume of CTL-CryoAB mixture prepared as specified above):
gently swirl the tube to permit complete mixing of the two media while the concentration of CTL-CryoAB slowly
increases.
• Aliquot the PBMC suspension into the pre-labeled cryovials: 1ml into each 1.8 ml vial.
• Place cryovials into Nalgene cryofreezing container, ‘Mr. Frosty’ filled with 2-propanol. The container should be at
room temperature.
† If Nalgene cryofreezing containers are not available, the following “low technology” method works equally well:
» Place the cryovials in a Styrofoam rack (e.g., racks in which the 15ml conical tubes are sold).
» Place a second Styrofoam container of the same type over the first one and tape the two containers together.
» Insert into a plastic bag leaving some air in the bag before taping it closed.
• Transfer the cryofreezing container (or bagged Styrofoam rack) into a -80°C freezer for a minimum of 12 hours. After
a minimum of 12 hours, transfer the cryovials into liquid nitrogen tanks for indefinite storage. Extended storage at
-80 °C will eventually result in decreased viability and recovery; therefore, transferring to liquid nitrogen within 2 days
is recommended.
Please note that the information contained in this document is privileged and confidential and protected from disclosure.
You are hereby notified that any dissemination, distribution, or copying of this document is strictly prohibited. © 2012. All rights reserved.
Cellular Technology Limited • 20521 Chagrin Boulevard • Shaker Heights, OH 44122-5350 USA
+1 216-791-5084 • +1 888-791-4005 Toll Free • +1 216-751-1373 Fax • reagents@immunospot.com • www.immunospot.com

Equipment and Reagents
EquIPMEnT: VEnDoR: CATALoG nuMBER:
Conical Tubes, 50ml, sterile Fisher 14-432-22
Serological Pipette, 2ml Fisher 13-678-11C
Serological Pipette, 5ml Fisher 13-678-11D
Serological Pipette, 10ml Fisher 13-678-11E
Pipette Aids, Drummond Fisher 13-681-19
Centrifuge, Allegra 6R Beckman Rotor Gh3.8A ALR6
Variety MicroPipette (20µl) Eppendorf 21-371-6
Pipette Tips (200µl) VWR 53508-783
Gloves Fisher 19-048-575A
Hemocytometer Reichert Bright-Line 02-671-5
Hematology Mixer Barnstead/Thermolyne 2-814-2
Vials, cryogenic, 1.8ml, sterile Nalgene 12-565171N
Nalgene Cryo Freezing Container Nalgene 15350-50
-80° Freezer Forma Scientific Inc. 55703-430
Liquid Nitrogen Storage Tank Thermo Forma 8030
Biological Safety Cabinet Forma Scientific Inc. Model 1286
REAGEnTS: VEnDoR: CATALoG nuMBER:
Whole blood Subject Source
Lymphoprep Greiner/Matrix 1114545
PBS sterile, Ca2+, Mg2+ free Cellgro 45000-436
Acridine orange (10mg/ml)* Fisher #A3568
Ethidium Bromide (1%) Fisher Biotech BP1302-10
CTL-Wash Supplement 10x Cellular Technology Limited, USA CTLW-010
CTL-Test Medium, 500ml Cellular Technology Limited, USA CTLT-005
CTL-Cryo ABC Freezing Kit Cellular Technology Limited, USA CTLC-ABC
RPMI-1640 Fisher Biotech BW12-167Q
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Please note that the information contained in this document is privileged and confidential and protected from disclosure.
You are hereby notified that any dissemination, distribution, or copying of this document is strictly prohibited. © 2012. All rights reserved.
Cellular Technology Limited • 20521 Chagrin Boulevard • Shaker Heights, OH 44122-5350 USA
+1 216-791-5084 • +1 888-791-4005 Toll Free • +1 216-751-1373 Fax • reagents@immunospot.com • www.immunospot.com