Hello Everyone.
Greetings,
I am planning to perform short term B cell culture (without any stimulation) from ovarian cancer patient (source of B cell PBMC and Ascites). I don't understand how long I should culture them. I will check the cytokines and Igs secretion by the B cell. Can anyone please suggest any protocol for short term B cell culture or any reference paper that can help me in my research. Thank you in advance.
Sincerely,
Razaul Haque
Hello @Razaul Haque
You can check this old post (https://www.researchgate.net/post/Can_anyone_share_recommendations_for_primary_B_cell_isolation_human_and_also_how_you_best_keep_them_growing_in_culture_over_time) or this ref (PMID: 27815447) but in both activation by interleukins is suggested to allow for growth in culture. Some suggest survival up to 2 weeks but extensive death and phenotype change may not fit your study check this past post too (https://www.researchgate.net/post/How_long_can_I_keep_freshly_isolated_human_PBMCs_in_culture_without_activation)
If they are normal B cells and not cancerous B cells (the cancerous ones could potentially grow on their own without stimulation), but normal B cells may only last 48h without additional stimulation before you start to see apoptosis.
I would suggest to take the B cells and re-stimulate them with PMA/Ionomycin in presence of Golgi transport inhibitors for 4-6 hours, then strain intracellularly for your cytokines of interest. B cells will also be producing intracellular Ig's to transport to the surface, so you can also perform intracellular stain for your immunoglobulin's of interest as well multiplexed with cytokines, and then run flow cytometry analysis. If you are not interested in short-term intracellular staining, perhaps overnight or 24h culture of the B cells in a low volume in 96-well plate, and then harvest culture supernatant for ELISA of cytokines and Ig's.
Steven Schutt Thank you so much for your kind answer. Actually, The sources of the B cells are PBMC and Ascites from ovarian cancer. I performed 24h culture without stimulation. But didn't satisfactory result. I don't know how whether I should increase the culture period or not. And I am interested to check the Igs by ELISA. If you have any further suggestion, I will be highly benefited. Thank you in advance.
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