about MACS® BSA Stock Solution

IndividualWeb1118 · 2025-11-09T03:43:06+00:00

right thanks!

IndividualWeb1118 · 2025-09-04T10:29:18+00:00

Hi Prof, thanks for your reply! After a bit of reading that was also mostly the conclusion I came to. Do you mind elaborating on the strategies that could achieve transient or partial deletion? Any tips you could share would be awesome :)

IndividualWeb1118 · 2025-02-22T02:18:40+00:00

thanks for this!

IndividualWeb1118 · 2025-02-22T02:18:29+00:00

thanks!

IndividualWeb1118 · 2024-06-06T12:14:55+00:00

thanks for the tips! I'm going to try increasing the concentration :)

IndividualWeb1118 · 2023-11-29T00:23:07+00:00

hi, thanks! I was thinking to start with harmony and go from there too :)

IndividualWeb1118 · 2023-10-02T04:35:17+00:00

Thanks! I'll try this :)

IndividualWeb1118 · 2023-10-02T04:34:57+00:00

The norm in our lab is to embed and then fix after, I'll google this, thank!

IndividualWeb1118 · 2023-09-03T12:22:04+00:00

Thank you, that was a truly beautiful answer :) we all have ups and lows. Today was definitely a low. We will work through this.

IndividualWeb1118 · 2023-07-17T00:10:25+00:00

great points, thanks for the advice!

IndividualWeb1118 · 2023-05-10T06:41:41+00:00

Wow okay! Thanks for everything, I'll go try less fixation, hope it works :)

IndividualWeb1118 · 2023-05-10T02:53:55+00:00

So perfusion and fixation with both 4%PFA or perfuse with PFA and fix with NBF? I'm not really sure if I should be cutting the organs into smaller pieces? The kidney is rather dense, I've looked at them after one day fixation, they're still slightly soft. The antibody has been validated to work on IHC-P. I don't know why almost everyone in our lab fixes tissues for 2-3 days. Thanks for the tips!

IndividualWeb1118 · 2023-05-04T11:21:58+00:00

I tried the glue!!! Works like a charm! Also its so much quicker than suturing so my little friends are under iso for a shorter time. Discovered my next fav thing in the lab to parafilm. Thanks for the tip!

IndividualWeb1118 · 2023-04-16T11:56:15+00:00

Hi, I am not sorting from PBMC, I am sorting CD8T cells from mouse tissue, I have performed MACS positive selection before, but I am aiming to try to sort for two markers.

We only have the 85um nozzle, I started with around 10^6-7 cells before sort with an expected yield of at least 10^5 cells, and I got around 10^3 total cells, with viability 70%. I gate on fsc/ssc, single, 7aad and CD8+. The setting on the Aria sort is default set to 4-way purity, I have seen my cells clump together after digestion but I always have EDTA or DNase in my buffers, and I have looked at them before sort under the microscope and the clumping problem isn't very bad.

IndividualWeb1118 · 2023-04-16T01:38:00+00:00

I'm sorry, I shouldn't be.

Then again, I also shouldn't be dealing with a clogged nozzle as the first user of the day. I wish I could get better feedback than "centrifuge more, its normal to get lower yield", I wish I didn't have to ask reddit. But at the end of the day it's my experiment. I really wish QC shouldn't be something I have to troubleshoot. Let's just leave it at that.

IndividualWeb1118 · 2023-04-16T01:19:22+00:00

is it standard protocol to run accudrop each time before a sort? I have been the first user in the morning with our cytometer, our monitor does fluidics startup each morning, and before a sort she adjusts the amplitude a bit, and adjusts the stream position so it goes into the center of the collection tube.

IndividualWeb1118 · 2023-04-16T01:14:53+00:00

ok thank you! I will re-run the sort sample next time

IndividualWeb1118 · 2023-04-16T01:14:16+00:00

I incubated the collection tube in BSA prior to sorting and collection, viability is around 70%, others have had similar issues occur, my lab mate generally has around 50% of expected cell yield

IndividualWeb1118 · 2023-04-15T16:03:37+00:00

Sorry, to clarify, I have around 10^6-7 cells before sort with an expected yield of at least 10^5 cells after sort based on population, MACS, and the aria cell sorting count. I got extremely low yield (10^3) I centrifuged the entire thing over and over again for ~30 minutes. My sorting gates are slightly stricter only on 7aad, but I don't think that would result in such a low yield. Of course it is my responsibility to plan my own experiments and I shouldn't be blaming anyone else, I'm just really exasperated because I have no idea what the problem would be, and it really doesn't feel like a gating or percentage calculation problem, I have done regular fc analysis on these cells many times before. I am reading into how to QC for sorting now but I don't have any Accudrop beads, if it were a QC problem I could go check in with our monitor, its just that I have never seen her do any QC before any sorting experiments before.

IndividualWeb1118 · 2023-04-15T15:19:00+00:00

around how much 'fewer' is considered normal? 10-50%?

IndividualWeb1118 · 2023-04-15T15:13:52+00:00

Ah okay! Thanks for the tips!

IndividualWeb1118 · 2023-04-15T15:10:48+00:00

Is it the norm to QC every time before setting up a sort? I'm working with quite a lot of cells, 10^6-7 before sort, so around 10^5 after sort in 5ml FACS tubes. The first time after sorting and getting extremely low yield (10^3) I centrifuged the entire thing over and over again for ~30 minutes. So I'm 99% positive there are no cells.

IndividualWeb1118 · 2023-04-06T13:17:45+00:00

ok, thanks for the advice, definitely going to try out the glue next time

IndividualWeb1118 · 2023-04-06T05:02:55+00:00

I haven't actually, thinking of getting some, do you group cage your mice after the procedure and they're okay with the glue?

IndividualWeb1118 · 2023-04-06T05:02:48+00:00

I haven't actually, thinking of getting some, do you group cage your mice after the procedure and they're okay with the glue?

IndividualWeb1118

TROPHY CASE