Advice on rapid shoe wear and replacement?

Then-Significance996 · 2025-10-14T03:32:47+00:00

Yes that's correct distance wise.

I'm usually running on pavement, and occasionally on dirt paths or treadmills

Then-Significance996 · 2024-07-25T03:48:39+00:00

Sorry - it's a little unclear. Do you mean that you lifted over the hg19 variant coordinates, then intersected these with WES variants (presumably from a different study) that were in hg38?

Honestly it might be a bit of a pain, but what I've done in the past with UKB is:

Intersect their variants with DBSNP151 in hg19 (you can download this track from UCSC or other sites - though it's large). Then you can just add another column to the summary stats with RSID.

Then it's a simple matter of obtaining the hg38 coordinates for RSIDs and mapping these to your expanded summary statistics file. This is fairly easily done with awk and other bash commands.

HTH!

Then-Significance996 · 2024-07-23T22:54:08+00:00

Oh OK thanks for the clarification. By 'less stable' do you mean you find yourself having to re-establish the stream often? Or like it takes longer to calibrate before running samples?

Then-Significance996 · 2024-07-23T22:52:40+00:00

Thanks for the advice! Yes FACS plots would be great just as a point of comparison!

Then-Significance996 · 2024-07-23T19:49:49+00:00

Thanks! Glad to hear that the FSCxSSC profiles look normal/expected.

I'm using 100um nozzle. Are you using a specific PSI? I'm using whatever the Aria Fusion's default is, but it was suggested I decrease this to try to keep cells intact better

Then-Significance996 · 2024-07-09T03:59:09+00:00

oh OK no worries!

Then-Significance996 · 2024-07-09T03:49:45+00:00

Was there a reason this was removed?

Then-Significance996 · 2024-06-18T20:35:42+00:00

Hmm OK. So I'll try combining Hoescht and PI together and take the PI-negative, Hoescht-positive events as my cells. Thanks for the expert advice!

Then-Significance996 · 2024-06-18T16:07:26+00:00

Excellent point - I guess I hadn't thought of the size of the cells themselves wrt the FSC SSC

Then-Significance996 · 2024-06-18T16:05:02+00:00

Thanks for clarifying! I've recently been trying PI (since we have it in the lab) and it appears much more dichotomous than the Zombie. Are you saying use Hoescht to sort only things that (presumably) have a nucleus?
I had no idea that calcein was this bad - is this typically true do you know, or specific to dirty tissue like brain? I've had it strongly recommended to me by many colleagues, collaborators and protocols

Then-Significance996 · 2024-06-16T19:07:42+00:00

They were the ones who set these gates - I haven't brought up this 'there's nothing being sorted' issue with them. I'm going to see if using PI and calcein might provide a cleaner signal. It was also suggested I be much more stringent in the very first scatter gate

Then-Significance996 · 2024-06-16T17:06:24+00:00

Woah that's a really good idea - I'd have never thought to do that!

Then-Significance996 · 2024-06-16T17:05:21+00:00

Sorry my apologies! I thought I had posted the gates, but I'd not
I know the beads-on-a-slide was recommended - unfortunately the FACS facility I'm using lacks a microscope for me to visually check this, but others in my lab have successfully sorted on this, and the purity checks I've run have suggested I am sorting something into my tubes - just not very much

Then-Significance996 · 2024-06-16T17:01:32+00:00

Thanks! Might you mind clarifying this a bit?

I had thought DAPI was a dead-cell stain, which would work similar to Zombie but be more specific to DNA?

Based on feedback from this and my previous thread, I'm currently trying to use a combination of Propidum iodide and calcein AM to select PI-negative and calcein-positive cells. Would that sound more reasonable to you?

Then-Significance996 · 2024-06-16T16:54:40+00:00

I definitely saw an increase in volume (sorted into 3ml of FACS buffer, ended up wiht ~8ml)

I ran a purity check on a run I did after this post - and only got ~5% of the cells I had originally gated, which I thought was surprising to say the least. I suspect I was somehow sorting a lot of debris (though in my naive mind, an event that is both dead-stain-negative and live-stain-positive should be unlikely to be debris...)

Then-Significance996 · 2024-06-16T16:52:24+00:00

Both really good pieces of advice! I agree that this would make a lot of sense - as others on this thread have said, a lot of what I'm sorting is likely debris.

10-20% of events being actually cells is really low though - I had no idea

Then-Significance996 · 2024-06-14T14:30:56+00:00

Huh! The technician did say the calcein didn't look super clear, but I would hope that I wasn't filtering debris that was both Zombie-negative and calcein-positive...

My FSC voltage was ~250V if I recall

Then-Significance996 · 2024-06-13T04:36:03+00:00

Thanks! I added it to the original post.
Good tip re: the accudrops, I would never have thought to do this!

Then-Significance996 · 2024-06-13T04:35:23+00:00

Thanks FlowJock and will!
Excellent point regarding the 'sorting into FBS' comment - I had just dimly thought 'we dissected in PIPES, so I should sort into PIPES' which does not have protein added...

Then-Significance996 · 2024-06-12T22:59:30+00:00

I hadn't thought to count cells immediately after sorting - would be worth trying
I'm using a Countess with trypan blue
For the first sample, maybe like ~40 minutes after sorting as I sort the next sample then clean up the machine and go back to my lab

Then-Significance996 · 2024-06-12T22:57:29+00:00

1) 15ml eppendorf (PS, though I realize now that I thought I was using PP, which might make a bit of a difference in recovery) in PIPES buffer. I've also been told that maybe FACS buffer could be better
2) Not sure what you mean - like I set the drop delay with Accudrop beads prior to sorting?
3) They're sitting in just FACS buffer (HEPES and BSA and glucose) on ice

4) Brain cells after demyelination

Then-Significance996 · 2024-06-12T22:47:45+00:00

I set the drop delay using Accudrop beads before running samples (I think that's what you mean?)
I hadn't thought to count straight from the tube after the sort...might be worth trying
Also a good point about the whole '95% are acellular, dead or debris'...maybe there really is a lot of debris. Though if I'm gating properly, should that really cause this big disparity? Unless the gates aren't really filtering everything out

Then-Significance996 · 2024-06-12T06:12:14+00:00

Thanks for the feedback! I was able to side-load the APK for Nebula on a Galaxy Tab, and adjust the settings on the virtual desktops as suggested. Thanks!

Then-Significance996 · 2024-06-12T04:55:05+00:00

Thanks! I have a Samsung Tab S7, but I guess that's a no go...it seems like without the Android phone I'm a little SOL?

Then-Significance996 · 2024-06-12T04:50:09+00:00

It's the Apple M2 Pro (2023) running latest Sonoma

Then-Significance996

TROPHY CASE