looking like erythrocytes

Is this picture taken right after PBMC isolation? Or is it a picture of the cell culture? If it is the latter, what day of culture is it? Also, what method are you using for PBMC isolation?

It could just be platelets, but I need more details to answer accurately.

Hi Jinu Yesudas ,

I agree with Zaven Karalyan

These could be red blood cells (RBCs) which are much smaller than PBMCs or simply shriveled dead cells. RBCs tend to sneak in during PBMCs isolation. Platelets are also another possibility. A better image with size indications or, as Mubasher Iqbal mentioned, more info on the PBMCs isolation and culture duration would help with a more accurate answer.

To get rid of the RBCs, use a RBC lysis buffer (if you don't have that on your protocol already)

Here are some protocols and a thread on the subject that may help.

https://www.kumc.edu/Documents/flow/Peripheral%20Blood%20Mononuclear%20Cell%20and%20RBC%20lysis.pdf

https://cdn.10xgenomics.com/image/upload/v1660261285/support-documents/CG000392_Demonstrated_Protocol_Isolation_of-PBMC_SingleCellRNA_Sequencing_Rev_A.pdf

Thread: https://www.researchgate.net/post/Can_you_add_an_RBC_lysis_buffer_after_PBMC_isolation

Best of luck!

Hello Jinu Yesudas

A buffy coat is a mix of lymphocytes, monocytes and platelets. The small structures that you see along with PBMC cells could be platelets.

You should get rid of platelets before you culture 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.

6. When most of the platelets are removed, resuspend PBMCs in complete RPMI 1640 containing 10 % FBS.

This protocol will help to eliminate most of the platelets.

Best.