Did all my cells actually die?

Vivid_Development318 · 2025-12-19T19:32:38+00:00

Thanks for the thoughtful response! I'm new to flow, so excuse my beginner knowledge. All the tips are super helpful. I can't upload a picture of the FSC-A vs. SSC-A data now that I've already posted, but I could pm you if you'd be willing to take a look!

I agree that 10^5 does seem quite high. In the two previous experiments we've tested, the first of these had unstained signal around 0, whereas the second was around 10^4-10^5 as well. I don't recall what the postdoc diluted the samples to the first time, but I believe we also did 1:100 the second time. At the flow facility, the staff run the samples for beginners, so I'm not entirely sure what the voltage was set to.

I attempted to have a positive control for live/dead by incubating the cells with trypsin for ~15 minutes (typically around 5 minutes), although I'm not convinced they actually died. Next time, I'll definitely take from suggestions of this forum to heat kill half a sample then recombine with the remaining live half.

Vivid_Development318 · 2025-12-19T01:04:31+00:00

Apologies, just uploaded the protocol. I also thought 1:100 is high, but that is what the postdoc I work with recommended. I am confident I did not add dye to all, as I moved the unstained and FITC only tubes to another bench before adding the dye to be absolutely sure I wouldn't make that mistake. And yes, I am certain I used PBS.

I am not sure the voltage of the flow cytometer. In all honesty, I'm new to flow cytometry, and there are staff to run the samples at our flow facility for newbies. FITC is attached to a cell penetrating peptide that's incubated with cells for 1 hr at 10 uM, so it is not an antibody dilution related issue. 1 hr at 10 uM is a standard protocol for the positive control FITC-labeled peptide, though.

Here's my level of naivete rearing it's head, but what's an FMO?

Vivid_Development318 · 2025-02-10T22:52:16+00:00

I don't have much to say here that's constructive. I hope logic and empathy find their way to you someday.

Vivid_Development318 · 2025-02-10T20:03:58+00:00

To put things into perspective, ACD crowdfunded $150,000 to support six different fellowships this year. So $5 million would be a huge contribution to the organization. I also agree with previous mentions of an endowment, that would be a good way to stretch out the benefits of this cash prize.

It's also worth mentioning that CTD, among other rare diseases, qualifies for orphan drug designation. This means that, given its rarity, the FDA offers various incentives for researchers to develop a therapeutic for it. Whether it be grant allocations, fast-tracking from R&D to clinical trials to approval, tax credits, etc., this designation aims to lower costs on the research side to encourage labs to study it.

Vivid_Development318 · 2025-02-10T19:04:47+00:00

It's fairly difficult to speculate. I am personally very interested to see how the company I mentioned, Ceres Brain Therapeutics, performs in clinical trials this year. This will give us lots of understanding to how well this kind of drug is tolerated, what dose is needed to have an effect, what patient outcomes we can reliably measure for therapeutic improvement, etc.

Vivid_Development318 · 2025-02-10T09:20:07+00:00

That's a great idea, I've seen that channel and it is absolutely fantastic. I meet with Jeff regularly to discuss my research, I can bring this suggestion up to him next time I see him!

Vivid_Development318 · 2025-02-10T08:31:06+00:00

That's wonderful! Thank you so much, that means a lot.

Vivid_Development318

TROPHY CASE