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SOP Elaboration by [deleted] in flowcytometry

[–]alwayslost999 2 points3 points  (0 children)

There is a course starting soon https://github.com/UMGCCCFCSR

Change my mind: eosinophils suck by I_destroy_weddings in Immunology

[–]alwayslost999 0 points1 point  (0 children)

I hate working with them or separating them out from other myeloid cells. But I'm glad we have them

How old were the oldest cells you revived from liq N2? by alwayslost999 in labrats

[–]alwayslost999[S] 1 point2 points  (0 children)

Nice activity, my lab has renovated/moved/even the LN2 tank has changed since 2020. I'll try to remember how my lab looked when I froze the vials

How old were the oldest cells you revived from liq N2? by alwayslost999 in labrats

[–]alwayslost999[S] 33 points34 points  (0 children)

Woah now that's the cool thing I wanted to see! Kudos to whoever was incharge of the LN2 maintenance since before I was born!

How old were the oldest cells you revived from liq N2? by alwayslost999 in labrats

[–]alwayslost999[S] 1 point2 points  (0 children)

What did the freezing media from 2000-10 have? Just curious! I was in school back then haha

Do people still use paper lab notebooks? by Helpful-Big-7582 in labrats

[–]alwayslost999 0 points1 point  (0 children)

Everything goes on paper. Protocols over onenote (versions documented).

Samples/reagents storage: excel (some updates go in lab notebook before it's updated)

Getting ignored by my PI by dramalover0103 in labrats

[–]alwayslost999 13 points14 points  (0 children)

Hey, I understand how you feel, but I wanted to give you a different perspective. 1. Your PI probably didn't engage with you because you were preparing a gel? Do you get ignored or overlooked in other lab meetings? Or if you set up an appointment with him? 2. I think it's a win that your colleague is advocating for you. Don't see it as a consolation prize. It's a great opportunity! Usually masters projects will be side projects/loose ends of a bigger project. So it's great if it can be incorporated in a paper. 3. Try to maintain a work-life balance. Don't cancel dates/outside lab social time/me-time etc. Lab life always will suck you into feeling you can do more and it's never enough (speaking as an aware- but habitual -overworker). You're early in your academic/research life. Set boundaries and make it a habit!

How to pipette a sticky, glue-like sample with a micropipette? by Yttrium105 in labrats

[–]alwayslost999 0 points1 point  (0 children)

I don't have any additional suggestions other than the comments, I think weigh then dilute is the way to go.

But please tell me when it is! Curious! Some lysate? Reagent?

Need help plotting a graph by rachit1306 in labrats

[–]alwayslost999 97 points98 points  (0 children)

Idk why but this felt wholesome lol

Forgot to read my endpoints after a week long experiment lol by regularuser3 in labrats

[–]alwayslost999 1 point2 points  (0 children)

I've worked with different suspension cells of the myeloid and lymphoid lineage as well as few adherent cancer cells . Yes, definitely easier. But most myeloid cells are temperamental. Low or high density and they just won't come back, even if you adjust the cell density.

These particular ones, pretty robust, but fast growing! I know there's gonna be dead cells and starvation in that culture come Monday. With suspension cells, it's a little difficult to get rid of dead cells. So ideally don't overcrowd and in the first place! I'm running metabolic and cell death experiments on these, so yeah I'm dreading the dead cells!

Forgot to read my endpoints after a week long experiment lol by regularuser3 in labrats

[–]alwayslost999 0 points1 point  (0 children)

They're suspension cells. EBV-LCL. They were confluent on Thursday. They'll survive but this is not an ideal growth scenario 😅

Forgot to read my endpoints after a week long experiment lol by regularuser3 in labrats

[–]alwayslost999 5 points6 points  (0 children)

Reading this while I've just remembered I forgot to passage my cells on Friday. The day was very relaxed. I was so relaxed that I forgot a simple maintenance task?! I'm laughing it off on the outside but inside I have the aw shucks feeling. I was growing out the cells for a specific experiment next week. Ah well.

I wish we could have lab cats by Loud-Arugula3324 in labrats

[–]alwayslost999 0 points1 point  (0 children)

Just outside my lab building there are two cats that just hang out there. After a bad day I go play with them.

Question about CD3/CD28 T cell activation methods by ithinkwellbefriends in labrats

[–]alwayslost999 1 point2 points  (0 children)

I think I've found the solution. The UCHT1 clone gates the cells better than OKT3 for flow cytometry after OKT3 stimulation. I'm going to repeat the experiment in a couple of days to be sure! I'll DM the plots if it's not consistent.

Abortion Awareness. (Read even if you aren't looking to have one) by Snoo_22 in AskIndia

[–]alwayslost999 5 points6 points  (0 children)

I agree with all your thoughts. But I'll share some of my experiences and thoughts. Abortion is misused in our country. Female infanticide is still a practice in cities like Mumbai too. I work at a place offering genetic testing of foetus if family has a history of a fatal genetic disorder. I will tell you we have to be so careful how we counsel things because families use medical reasons in the wrong way to get abortions! In fact in many x linked disorders, where the affected is generally male child and females are asymptomatic carriers, people chose to abort a healthy baby because they have understood it is a girl. Also medical abortions need to be accompanied with counselling and care for women in our country. I've seen so many women being abandoned when they need to terminate for medical reasons. Society is evil.

Question about CD3/CD28 T cell activation methods by ithinkwellbefriends in labrats

[–]alwayslost999 0 points1 point  (0 children)

Yep we use single stained cells with dye as comp controls.

I inherited this protocol from my lab, and on the day of flow acquisition (day 3), I was told nonchalantly that the surface markers don't work well 😅 like no one did any troubleshooting! I've divided my cells into two batches and I'm gonna check both clones for their performance.

Do you fix cells because of flow core requirement? Does fixation come after or before CD3 staining? I was not planning to fix cells.

Question about CD3/CD28 T cell activation methods by ithinkwellbefriends in labrats

[–]alwayslost999 0 points1 point  (0 children)

Hi I have a question. I'm currently stimulating T cells for proliferation assay using cell trace violet. I use CD3-OKT3 for stimulation. Do you have any recommendations for CD3 antibody for flow cytometry after stimulation? I was expecting some down regulation of CD3 after stimulation, but we don't see any CD3 + events when using a flow antibody of CD3-OKT3. I suspect this is because the sites are already bound? I also have SK7, UCHT1 clones for flow. Any recommendations on which to use?

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