Hello Paula K. Sato

Yes, you can. PBMCs from frozen buffy coats would become necessary when there are studies with elevated number of participants and for sample collections in multi-center studies.

You may follow the below protocol.

1. Centrifuge the collected blood at 2000 x g for 15 mins at 4 deg C in a swinging bucket rotor, switching-off the centrifuge brakes.

2. Collect the cloudy buffy coat (the layer between the upper plasma phase and the lower phase containing most of the erythrocytes), containing the enriched PBMC fraction into a cryovial tube.

3. Resuspend the buffy coat in freezing medium containing 90% FBS and 10% DMSO.

4. Freeze the vial in a controlled rate freezing container at -80-degree C. For longer storage, you may store cells in liquid nitrogen.

5. During thawing of buffy coat, add the same volume of PBS to the thawed buffy coat.

6. The diluted buffy coat is gently layered on Ficoll (centrifuged at 3000 rpm, 20 minutes, refrigerated centrifuge, swing rotor, break off).

7. Collect the PBMC layer.

8. PBMCs are then washed with 5 ml PBS two times.

Best.

Malcom Nobre, thank you for your answer! I’ll follow your recommendations!