Quick guide to PBMC freezing

Tips & Guidelines

Sahra L.
By Sahra L.    time to read 2 min

You nailed it!

The long-awaited blood sample you need has finally arrived! You extracted PBMC and managed to obtain a great yield, albeit with a little shivering…

Perfect, half the job is done.

Now, the plan is to carry out a thousand experiments with THIS precious sample. But there are only 24 hours in a day, and unless you’ve found a way to duplicate yourself, you’re going to have to come up with something.

Widely used in laboratories, freezing cells is a good solution. But improper freezing can just be a waste of your previous effort. To help you succeed, we prepared some guidelines and tips for PBMC freezing!

Get the complete guidelines

Francisbio happy

Count cells as a start

pellet and resuspend cells in FCS at RT
Malassey cell also called malassey counting chamber used to determine the number of particles per volume unit of a liquid

The first step after purification is to pellet and resuspend cells in FCS at RT.

Then count the cells to dilute at a proper concentration.

To count cells, use a small tool called a “Malassez counting chamber”, or a hemocytometer. Invented by Louis Charles Malassez, this tool is a must-have for every lab. It may cost several hundred dollars and the glass to be put on it is particularly fragile, so make sure to treat it gently…

Pipet 10µL of your resuspended cells in the chamber, count them, and appreciate the round shape of happy cells. The magic formula for calculating the concentration in 1 ml is the following (you’re welcome):

Cells per mL = Average number of cells per square * dilution factor * 104

Final step is aliquoting!

Now you are ready to dilute cells with FCS containing DMSO.

Tips: consider that about 50% of the cells will die during the freezing process. Therefore, to obtain a concentration of 10 million cells per ml after thawing, prepare a stock of 20 million cells.

Aliquot 1 ml of the solution per cryotube. Store at -80°C for 24 hours, transfer in liquid nitrogen, and you’re all set! Frozen PBMC can be stored for approximately… an entire lifetime!

But good freezing is pointless without proper thawing

Slowly thaw cells in a water bath at 37°C and transfer them to a flacon containing RPMI. Centrifuge and discard the supernatant, and then resuspend in medium to rinse cells.

The cells are now ready for your assay!

dilute cells with FCS containing DMSO
Cells aliquoting to cryotube process
Application note - How to freeze PBMC

Guidelines for PBMC freezing

…and more tips to set up your experiment

Download technical note