Cryopreservation of PBMCs - FAQs

November 9, 20092comments



C ryopreserved Peripheral blood mononuclear cells (PBMCs) are a common specimen source for studies of immunological responses to vaccines, immunotherapies, etc. The health and viability of cells recovered post-cryopreservation are of course critical to the success and accuracy of immunological assays performed on them. Studies have previously shown that the results of functional assays are strictly dependent on the viability of the cryopreserved PBMC.

The utility of cryopreserved PBMC for functional immunological assays is predicated on their viability and functional ability in vitro. The use of cryopreserved PBMC in immunological assays poses challenges, including the availability of adequate equipment and the need for technical proficiency. Assays must be adapted and validated for the use of cryopreserved PBMC, and the quality of the frozen cells has to be monitored to ensure reliable results in functional and phenotypic assays.
Frequently asked questions in cryopreservation of PBMCs:

Are Ficoll-Hyopaque and Histopaque-1077 the same?
What is the difference between Ficoll overlaying and underlayering technique?
Why do I have to remove the supernatant - HBSS-Plasma layer?
Why I can't remove all the density gradient?
How much supernatant should I leave before isolating the PBMC band?
Should I remove the cells stuck to the side of the test tube?
What should I do if I accidentally mix the layered blood with ficoll?
Why do we need to use a centrifuge with swinging-bucket rotor?
Can a vertical rotor be used with Ficoll?
What temperature should I use during centrifugation?
What speed and time should I use in my centrifuge?
What should I check if I do not get a PBMC layer?
How much buffer do I need for my cell wash and why?
What is the total number of cell washes?
How much buffer is left in my test tube after I decant the supernatant?
What volume of buffer should I use to re-suspend my pellets?
What is the Forward pipetting technique?
What are the effects of how I pipette?
What are some common pipetting technique errors?
What type of plasticwares should I use?
Are powdered gloves acceptable for processing PBMCs?
What is the pH, solution storage requirement and length of time that cells can stay in trypan blue?
What are some common problems with a Hemocytometer?
How to perform an accurate count?
What are the formula for cell counts?
What is the purpose of using DMSO?
How much DMSO do I need to add to cyropreservation media?
Cooling rate for Mr.Frosty and Rate Control Freezer
How do we know a reagent is stable?
Causes for unsatisfactory performance?

Click here for a presentation that answers all these FAQ...
Source: Tania Garrelts/Raul Louzao,Duke University School of Medicine, IQA Center

Keywords: PBMC, Cryopreservation, Peripheral blood, LN2 storage, Cryo, PBMC isolation, DMSO
Share this article :

+ comments + 2 comments

December 7, 2010 at 12:05 AM

Thank you for a very useful presentation. But I can't see the videos attached to it =/

December 7, 2010 at 3:29 PM

Thanks Shyne...

Pls go to https://iqa.center.duke.edu/modules/iqa_plabs/index.php?id=26

and run the powerpoint in your browser without downloading the ppt and you can see the videos.

Post a Comment

 
Support : | Mas Template
Copyright © 2013. iNVISIBLE YET iNVINCIBLE - All Rights Reserved
Published by Mas Template
Proudly powered by Blogger