I want to investigate calcium mobilization of lymphocytes (B cells and T cells) upon receptor ligation. I have purchased some Fluo-4 AM to do this, and when researching possible staining protocols I noticed that everyone is using different buffers (e.g. HBSS, RPMI, HEPES). Could you please tell me which buffer(s) have worked best and which ones to avoid?
I'm not entirely sure it matters, so long as you keep conditions uniform across all treatments and compare to good positive and negative controls. I, too, have noticed odd preference in protocols for things where they logically should not have a bias against using normal medium. It seems to me that there is one of three possible reasons for this: (1) there legitimately is a reason to use one buffer over another; (2) they are doing some other key experiment in their study and want to use the same medium across all experiments; (3) the lab has an internal preference for some buffer (usually HBSS or similar) for imaging.
I agree with Dear Yaron, we have have used RPMI1640 medium, as we maintained our cells in this medium. I have attached below our protocol, if helps!
Cells were initially equilibrated at 37uC for 45 minutes with loading medium containing Fluo-4 AM (2.5 uM), pluronic acid F127 (0.02%) and sulfinpyrazone (0.25 mM) in RPMI 1640 medium. After de-esterification of Fluo-4 AM, cells were washed with RPMI1640 medium containing sulfinpyrazone (0.25 mM, 37oC,45 minutes).
I have also attach our paper here, if helps!
Thanks, Good Luck!
Article Malabaricone-A Induces A Redox Imbalance That Mediates Apopt...
In the experiment we conducted with neutrophils, we did incubation of the cells with Fluo4-AM in RPMI or DMEM containing 10% fetal bovine serum for 30 minutes at room temperature. After incubation the cells were washed and resuspended in reading Krebs containing 0.1% BSA.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology