u/matchaduo

hi! i'm an undergrad working on panel design for my tfh-b cell co culture with some antibodies we have in lab. I work with a spectral flow cytometer (novocyte opteon). also im planning on using an Fc block but I'm not sure if i should also look into the True-Stain buffer also.

i have the following

Live/Dead: Near IR 780 (works great!)

CD4: AF700

CXCR5: PE/Cy7 (works great!)

ICOS: BV750 (works great!)

CD19: BV650

CD27: AF488

CD38: BV421

IgD: PerCP-Cy 5.5

I was wondering if this would work well since I know PerCP-Cy5.5 is a bit finicky but IgD should be highly expressed! Any feedback would be appreciated!

hiii everyone, i’m flying back with Alaska with a 36x42 poster from my internship and it’ll most likely be in a poster tube. I’m also bringing a carry on and a personal backpack so I’m worried that the tube won’t be accepted because it’s oversized or might be counted as a third item. does anyone have any experience with a poster tube? thanks!

hi! so my original flight was cancelled so delta rebooked me for the next day. I originally booked main classic so I picked my seat ahead of time but after being rebooked it now says seat assigned at gate. I’m a bit concerned that this flight is overbooked since I have an internship scheduled for the next day and have to be on this flight. does anyone have any experience? thanks for your help!

hii everyone

i've been having some weird issues with low events and was wondering if anyone had any insight.

i usually have around 0.3M human T cells per tube and normally acquire around 100,000 events on a Novocyte Opteon. However, the last two runs I've been getting 1k-5k events per tube. I'm very careful when aspirating and I see a visible cell pellet throughout my washing steps for surface and intracellular staining. i also think it's very unusual to have aspirated the pellets out for all my samples for 2 experiments in a row when i have been working with this for a few years.

my compensation looks normal and the only thing I can think of is that I switched to filtered pipette tips although I doubt that would cause a dramatic drop in counts. i try to limit loss when transferring from my plates to tubes. I checked under the microscope too and my cell counts looked good. any insight would be appreciated!

hi! i'm an undergrad working on human tfh-b cell co culture and would appreciate any feedback on my panel or if anyone has any suggestions for antibodies. i currently use a spectral cytometer (4 laser novocyte opteon: violet, blue, yellow, red) and put everything in a spectral viewer. i tried to design it in a way where lower expressed markers were paired with brighter fluorophers. Near IR 780, PE/CY7, BV750 have worked great together on previous experiments. i'm looking to evaluate Tfh phenotype, B cell differentiation, class switching, and plasmablast formation. thanks for your help!

Proposed Panel:

Live/Dead: Near IR 780 (currently used)

CXCR5: PE/CY7 (currently used)

ICOS: BV750 (currently used)

CD4: AF-700 (also could potentially switch to APC, BV605)

CD19: BV650

CD27: FITC

CD38: BV421

IgD: PE

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!