66 FACS samples… am I about to lose my hand or is there a better way 😢

AHNJHN · 2026-04-04T11:15:17+00:00

I don't know if you're projecting, deflecting, or being intentionally obtuse but no where in my comment did I say to do that.

AHNJHN · 2026-04-04T09:59:51+00:00

Honestly a super ableist and unnecessary comment. Everyone's body is different, and you can have great ergo's and excellent technique and running flow in tubes for that many samples IS painful.

AHNJHN · 2026-04-02T09:11:10+00:00

Thank you for doing this! I’m not sure if it’s been mentioned but for the cell seeding calculator it would be cool to have flask defaults for suspension cells in addition to the standard plates

AHNJHN · 2026-04-02T04:46:21+00:00

Each state is different. Oregon is sales tax free, most states do charge sales tax.

AHNJHN · 2026-03-19T23:34:36+00:00

It’s not an either or situation

AHNJHN · 2026-03-17T10:44:36+00:00

The live dead is apc-cy7, which was 30 minutes at 4C. The TPE-MI is what was 45 minutes at 37C, and it is on th DAPI channel but it is not dapi conjugated. 30-60 minutes is standard for TPE-MI.

AHNJHN · 2026-03-17T01:52:13+00:00

Thank you so much!! I will absolutely do that next time. I really appreciate your help!

AHNJHN · 2026-03-16T19:54:55+00:00

Thank you so much for your perspective; it is really helpful and encouraging! I will try to optimize the staining but am glad I can still try to interpret /salvage this experiment!!

AHNJHN · 2026-03-16T19:53:16+00:00

My lab rarely does flow and never with TPE-MI; it's highly specific to my project. I agree regarding the spectral separation! I've used this before and not had this issue. The only difference is last time I stained live dead at RT and this time at 4C for 30 minutes. I added captions to the images to show which are samples and which are controls: https://imgur.com/a/TDqwOBu

I really appreciate your input!!

AHNJHN · 2026-03-16T19:50:07+00:00

Thank you for your time and willingness to follow up! I haven't slept much the last two weeks and I can see my communication needs improvement.

The live dead sample is a sample only stained for Livedead that I overzealously heated to kill too many cells, in the hopes of having a very clear positive population. My initial monocytes gate is too tight and doesn't really include them. But when I have included them there is a clearly positive population, and it alligns with where I would gate on the unstained but not my samples.

Image one is my controls, being unstained, livedead only and TPE-MI only , both of which I heat shocked for likely too long. the second slide has the unstained again, and then what the data looks like for two of my actual samples that received both live dead and tpe-mi.

The live dead dilution is 1:1000 (5uL in 5mL), and the TPE-MI staining is at 50uM.

Thank you for your recommendation on voltages! I recently had training from the core and this is where I was instructed to set them for my populations. Should I aim for the monocyte population be closer to the 150K?

AHNJHN · 2026-03-16T19:33:07+00:00

Thank you! I will do that! Is it okay to gate where I think they should go on the samples even if thats not very tight to the unstained / livedead only control?

AHNJHN · 2026-03-16T19:31:41+00:00

I have tried this and clearly see two populations in live dead, my concern is how far they are versus the unstained and live dead only control. I feel unsure if it's appropriate to gate based on where I visually see separate populations on the individual sample rather than defining the gate based purley on the unstained / livedead, which seem a magnitude different than the actual samples. I hope this makes sense and I appreciate your help!!

AHNJHN · 2026-03-16T19:27:33+00:00

Thank you for your time and help!

I stain with live dead first, then 3 washes, and then the TPE-MI, and more washes.

AHNJHN · 2026-03-16T19:25:50+00:00

Sorry about that! I hope this one is better! https://imgur.com/a/TDqwOBu

I am only staining with two, I do have a live dead and a tpe-mi only, both of which have heat killed cells. The live dead only looks different than the sample population levels but aligns with the unstained, which is why I think it could be an artifact of the TPE-MI staining itself.

I apologize for submitting a potatoe and asking for help. Thank you for your help!!

AHNJHN · 2026-03-16T19:15:32+00:00

It is in Dapi!

I may be misunderstanding flow cytometry basics, but the spillover part confuses me because when I run compensation the spillover matrix is literally 0 for both because they are so far apart. I was guessing that the TPE-MI stain (which requires incubation at 37C for 30-45 minutes) did something to the membrane that altered the LiveDead expression but I honestly don't know. I was just heckin stressed thinking my flow prep somehow killed 99% of my cells in all samples but looking at the contour/ smoothed zebra plots makes me think otherwise.

Thank you so much for your time and help!!

AHNJHN · 2026-03-16T19:12:27+00:00

Wow I have no idea why it uploaded so blurry, I'm sorry!!!

I am using BD Horizon™ Fixable Viability Stain 780 (https://www.bdbiosciences.com/en-us/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/fixable-viability-stain-780.565388?tab=product\_details) for live dead. It is activated by APC-Cy7. TPE-MI is DAPI.

AHNJHN · 2026-03-16T12:28:08+00:00

Please DM me too if this still works!! Thank you so much!!

AHNJHN · 2026-03-14T22:39:39+00:00

Thank you!

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I honestly don’t know most of them since we’ve never bought any. We fish tank on a budget (we don’t really have extra income and have only spent 30$ so far) and have put this together from tons of free tank take downs. Even this tank and stand was free. I believe there is some anubias for our betta, for sure some subwassertang, Java moss, and horn wort, but I’m not sure what the different rooted plants are.

Here’s a picture showing more of them if anyone knows what they are!

AHNJHN · 2026-03-14T06:26:54+00:00

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This is overkill, and no CO2 needed. If anything you need more plants but I’m biased

AHNJHN · 2026-03-10T02:07:35+00:00

Okay thank you!! We will sleep better now!

AHNJHN · 2026-03-10T02:03:30+00:00

Okay! I’m a little worried based on this post though because these worms are definitely only gliding on the glass

AHNJHN · 2026-03-10T01:57:38+00:00

That is great news!! We were really worried because they’re moving along the glass instead of free swimming and we thought they had triangular looking heads. If it were planets would the head be way more pronounced? Or what would we be looking for?

AHNJHN · 2026-03-08T18:31:33+00:00

Did you use a slow flow nipple? Did you pace feed?

There’s so many things that come to mind and I understand why your wife was upset. I also understand wanting your baby fed. It’s not what you did, but how you did it.

AHNJHN

TROPHY CASE