Did you check the media?

I am using RPMI+10% serum +antibiotics

Thanks for reply my quest.

Hello Mayra Silva-Miranda

Cell clumping is a common problem following thawing of PBMCs. You may want to refer to the article attached below.

Article DNAse treatment following thawing of Cryopreserved PBMC is a...

This problem can arise because of the following.

1. Thawing is a stressful process. This stress can accelerate the rate of cell death resulting in the release of sticky DNA molecules from the dying cells that can clump neighboring cells together.

2. If PBMCs are contaminated with platelets during the isolation process, then you are likely to get aggregates.

Therefore, incorporation of a DNase treatment step in the standard thawing procedure would be necessary to avoid clumping. Once removed from liquid nitrogen, the vial containing PBMCs should be placed in a 37 deg C water bath until they are almost completely thawed, then transferred to complete media of choice and centrifuged at 300 x g for 10 minutes. Carefully remove and discard as much of the supernatant as possible, taking care not to disturb the cell pellet. Gently tap the tube to resuspend the pellet.

Add DNase I to the cell suspension to yield a final concentration of 100 μg/mL. Add DNase I solution dropwise to the cell suspension while gently swirling the tube. Incubate at room temperature for 15 minutes. Wash the cells with culture medium containing 2% FBS. Gently invert to mix, then centrifuge at 300 x g for 10 minutes. Discard as much of the supernatant as possible, then gently resuspend the pellet. This will help in preventing cell clumping.

As you know that a buffy coat is a mix of lymphocytes, monocytes and platelets. There is a high probability that PBMCs may get contaminated with platelets.

You should get rid of platelets before you freeze PBMCs.

You may follow the below protocol.

1. Perform a standard density gradient centrifugation.

2. Pipette out and discard the plasma layer before isolating PBMCs.

3. Transfer PBMCs to a new tube. Wash the isolated PBMCs in PBS containing 2% FBS.

4. Centrifuge for 10 mins at 120 x g with brakes off. Carefully remove and discard the supernatant.

5. Repeat the wash process two more times. This will help to get rid of most of the platelets.

Kindly note that if PBMCs are isolated from patients, then there is a high probability that there could be a change in the biophysical property of PBMCs under certain pathological conditions because of which you may observe clumping. For instance, in blood disorder such as chronic myelogenous leukemia (CML).

Best.

thanks. Is a great information. I will try it.