Thawing Protocol for Cryopreserved Human PBMC - Cellular ...

Thawing Protocol for Cryopreserved Human PBMC
Page 2 of 3
• L-glutamine supplemented CTL-Test Medium should always be added warm (37 o C) to cells. Ideally, the medium should
be placed into a CO 2 incubator in a tube with lose cap for 20 minutes to warm up and for the pH to reset itself. (CTL
media are buffered. Slight fluctuations in pH might occur, resulting in fluctuation of color.)
• Each PBMC cryovial will yield between 10 and 20 million PBMC. If, e.g., the PBMC are to be plated at 4 million/ml
(400,000 per well), prepare 5ml of CTL-Test Medium for each vial of PBMC thawed.
D. Thawing of the cryopreserved PBMC
• Warm up CTL Anti-Aggregate Wash Solution (20ml ) and CTL-Test Medium (5ml) by placing tubes with lose cap into
37°C CO 2 incubator for 20 minutes. Both media should be at 37°C and CO 2 equilibrated when added to the cells. Raise
the temperature in the cryovial that contains the PBMC rapidly to 37°C. It is recommended to use a 37°C sand or glass
bead bath (available from CTL). Using a 37°C water bath is adequate, but it increases the chance of contamination.
Under both conditions, the temperature should rise to 37°C in 10 minutes.
• Aseptically transfer the cells from the cryovial into the 50ml conical tube that has been pre-warmed in a 37°C water
bath. (At this point, the contents of up to 4 cryovials from the same sample can be pooled.) Pour cells gently into tube
to minimize shear forces and air bubbles.
• Rinse each emptied cryovial with 1ml of warm (37°C) CTL Anti-Aggregate Wash Solution to recover residual cells.
Slowly, drop-by-drop, add the rinse to the cells in the 50ml tube while gently swirling that tube. Pipette the rinse gently
because sheer forces will induce apoptosis in the cells. Subsequently, add slowly (over 1 minute) more warm (37°C)
CTL Anti-Aggregate Wash Solution, diluting the content of the cryovial to ~6x of the original volume (e.g., when
thawing a single vial, final volume should be 10ml).
• Centrifuge cell suspension at room temperature at 330g for 10 minutes with rapid acceleration and brake on high.
• Decant the supernatant, and carefully resuspend the cell pellet by tapping the tube (avoid pipetting or Vortexing). Add
10ml warm (37 o C) thaw solution for each PBMC vial thawed.
• Count the cells under a UV microscope with Acridine Orange/Ethidium Bromide for the most accurate results
(counting with Trypan Blue can overestimate cell counts by including apoptotic cells and erythrocytes). Precise counting
requires that also the numbers of apoptotic cells is established since such cells are still alive, but will be dead before
the functional assay is completed (inquire with CTL for dyes that permit staining of apoptotic cells for counting by
UV microscopy. Please be advised that the use of some automatic cell counting devices can lead to a high level of
inaccuracy in the counting results if they are not calibrated and adjusted in direct reference to manual counting of
freshly thawed samples. We advise to verify viability first by visual assessment until all the machine generated results
have been verified. Viability should be over 80% (typically, it’s over 95% for CTL’s ePBMC ® cryopreserved PBMC).
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