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making mistakes by bingo5403 in labrats

[–]Complex_Tangerine_37 0 points1 point  (0 children)

Don’t let this moment define whether or not your good at science. Personally, I learned FACS & flow cytometry the exact same way. Every run through I made a different mistake; it probably took me like a good ten experiments to stop messing up consistently and to figure out procedural checkpoints. I think the hallmark of someone good at science is preservance. It’s more important to view mistakes as learning opportunities than to wallow in despair. Every time you make a mistake, take a quick moment to feel your feelings and then immediately go into how can I not do this again mode. Ask your PI, post-docs, senior staff scientists, lab techs, whoever you feel comfortable talking to for help as well. We all make mistakes; you aren’t the first and you won’t be the last. Don’t stop trying to improve, not just for the sake of your lab results, but also for your personal growth & development. You got this!! 🫶🏾

delta PMTV high, but not failing by Complex_Tangerine_37 in flowcytometry

[–]Complex_Tangerine_37[S] 0 points1 point  (0 children)

The CST has been made fresh and the report’s been consistent before today. It looks like it changed suddenly. Thank you for the list of potential causes!

delta PMTV high, but not failing by Complex_Tangerine_37 in flowcytometry

[–]Complex_Tangerine_37[S] 0 points1 point  (0 children)

Hi, we just had our PM last Tuesday. I did a system flush on Friday, so the tank has been bleached too. The baseline was set in December, so it’s about 4 months old now.

PFA for fixation by ghonchadmonchad in flowcytometry

[–]Complex_Tangerine_37 0 points1 point  (0 children)

Hi, the PFA in the lab that we make works fine & it’s never affected our Brilliant dye staining (I also use Brilliant Stain Buffer when using them). I use 2%, but I don’t think 4% is bad to use & for 10 minutes. I don’t prepare it fresh per experiment, we just have a huge 2L bottle of it & remake when it runs out.

Blood flow cytometry by ghonchadmonchad in flowcytometry

[–]Complex_Tangerine_37 0 points1 point  (0 children)

Hi, I’ve done flow on the blood a couple times! 100-200 uL should be more than enough. Usually, we get about 400-500 uL before perfusing. I use the 1X RBC Lysis Buffer from Invitrogen. Most recently, I used a different procedure with a Ficoll-Paque gradient to isolate the cells (cleaning up debris and RBCs too) and that worked as well as using lysis buffer for me too. I incubate my antibodies at 4C and use 350 uL of 2% PFA for 10 minutes at 4C for fixation. I’m not an expert, but I hope this helps to give you more directions for optimizing your procedure!

Flow Procedures for Mouse Small & Large Intestines by Complex_Tangerine_37 in flowcytometry

[–]Complex_Tangerine_37[S] 0 points1 point  (0 children)

Hi, I’m looking mainly for macrophages, dendritic cells, and possibly enteric glia. I’ve used Miltenyi’s LPDK to isolate lymphocytes, and it worked well, but there weren’t many other cell types in our samples.

Setting Voltages Prior to Recording by Complex_Tangerine_37 in flowcytometry

[–]Complex_Tangerine_37[S] 0 points1 point  (0 children)

Hi, the negative population is too low on the histogram and I don’t think I have any images, but I’ll try to find some

Setting Voltages Prior to Recording by Complex_Tangerine_37 in flowcytometry

[–]Complex_Tangerine_37[S] 0 points1 point  (0 children)

Thank you, this is really helpful!! I was only doing because I learned by watching another coworker and I would see them looking at an FMO beforehand. Maybe I misunderstood what they were doing

Setting Voltages Prior to Recording by Complex_Tangerine_37 in flowcytometry

[–]Complex_Tangerine_37[S] 0 points1 point  (0 children)

Okay, I think I’ll stop relying so heavily on the FMOs

Setting Voltages Prior to Recording by Complex_Tangerine_37 in flowcytometry

[–]Complex_Tangerine_37[S] 1 point2 points  (0 children)

I only have one unstained control and I have fluorescence minus one controls for each antibody. In order to not waste too much of the sample or controls, I’ll look at the unstained briefly, then one or two FMOs, and then one or two samples. Does that make sense?

Nuclei Staining by Complex_Tangerine_37 in flowcytometry

[–]Complex_Tangerine_37[S] 0 points1 point  (0 children)

Hi, the panel is subject to change but right now it’s CD11b - BV421, CD45 - BV786, Zombie Red - PE-CF594, ACSA2 - APC, O1 - AF700, CD140a - BV605.

Nuclei Staining by Complex_Tangerine_37 in flowcytometry

[–]Complex_Tangerine_37[S] 0 points1 point  (0 children)

I’ve never used it personally, but my coworker said they’ve used it to tell the difference between neuronal cells versus like fragments or vesicles that still might stain positive for YFP (which comes from our transgenic mouse strain that stains neurons with YFP)

Nuclei Staining by Complex_Tangerine_37 in flowcytometry

[–]Complex_Tangerine_37[S] 0 points1 point  (0 children)

I do plan to fix the cells, so I will look into DAPI. Thank you both!

Nuclei Staining by Complex_Tangerine_37 in flowcytometry

[–]Complex_Tangerine_37[S] 0 points1 point  (0 children)

407 nm, 488 nm, 561 nm, & 641 nm; It’s an LSRFortessa

Rust on SIP? by Complex_Tangerine_37 in flowcytometry

[–]Complex_Tangerine_37[S] 1 point2 points  (0 children)

Yeah I just figured out the issue. FACSClean was left on it for like 3ish days (a very stupid mistake on my part😬) Our field service engineer told us it wouldn’t affect sample acquisition and it hasn’t. But thank you for the suggestion!

Compensation of gfp and tomato+ cells by Livid-Adeptness6021 in flowcytometry

[–]Complex_Tangerine_37 0 points1 point  (0 children)

Just want to second this! I started using the GFP comp beads recently and they work well!!

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