u/matchaduo

Hi! Has anyone used the biolegend stimulation conditions for IL-21 for human T cells (ionomycin 50 ng/mL and ionomycin 1 ug/mL) for ELISA/multiplex assays?

I was planning on stimulating without brefeldin A for an extended amount of time like 16-18 hrs but I am considered if those concentrations are a bit high.

Does anyone have any suggestions since literature seems to provide conflicting information.

For context:
I work with human in vitro polarized Tfh cells and I’ll need to freeze supernatant in the -80 from a very large amount of cells for downstream analysis so I unfortunately won’t be able to troubleshoot conditions.

Would appreciate any insight!

hi! so i’m doing Tfh-B cell co culture. One of my gates would be CD4 vs CD19.

I currently have BV650 for CD19 in lab and a few CD4: options available: BV605, AF700, APC.

I was leaning towards CD4(AF700) and CD19 (BV650) and was wondering if this would provide the most efficient separation for clean downstream gating? I’m on a spectral cytometer and any insight would be appreciated! Thanks!

hi! I’m going to be lysing my differentiated suspension T cells with qiagen RLT for RNA extraction and I’ve seen conflicting information on whether I should directly lyse after pelleting and removing supernatant or do a cold PBS wash first. Any insight would be appreciated!

hi! I have a few questions about panel design and would appreciate any insight! thank you so much!

1. I currently use PE/Cy7 (CXCR5) and PE/Dazzle 594 (BCL6) in the same panel and it’s worked great. I wanted to add IL-21 (PE) in since we have that in lab but I believe that’s not ideal??? I’m considering APC instead??

2. BV605 and Pe/Dazzle 594 overlap but since they’re excited by different lasers would it be fine?

3. Does anyone have any experience with BV421 (FOXP3) or have any recommendations for any other bright antibodies other than PE?

thank you again!

hi! my lab has a few vials of unopened dynabeads that have been expired for a little over half a yr. does anyone have any experience with T cell activation quality with expired beads. I’m really hesitant to discard since they’re so expensive 😭😭😭😭. i think the best way to test it out is to try them out myself but I’m worried about wasting cells. thank you so much!