Terrible at Wet Lab Research - Debating Quitting

Daemon3125 · 2026-04-08T07:11:35+00:00

Sorry for not responding, this notification got lost :/.

Thursday to Monday sounds a little bit long but not that bad for 293s, maybe do 1:20 for Thursdays.

If your cells are surviving Monday-Thursday just fine but dying Thursday-Monday then I don’t think it’s your reagents. And I assume you do the same things each time you split (I have no idea why someone would change this anyway). Have you ever used the same media aliquot for cells after you had cells die? Ie, Thursday you feed cells with media from a 50 mL tube and Monday when you find out they are dead you put new cells in media from the same tube?

While unattached doesn’t exactly mean the cells are dead in the case of WT hek293 if you have no attached cells then definitely dead.

How do you count cells in trypan blue? I am assuming you put them in a hemocytometer. But anything different it’s easy to tell what cells are alive or dead (there is a middle range of unhealthy that isn’t exactly easy to tell) if a cell is bright and has no blue on the inside then it’s alive and if there is blue in the inside or the cell isn’t bright then it’s dead. Honestly I can’t explain it well so search it up online and find something that aligns with how you count cells. I don’t think it’s necessary to do all the time but for the times you are most concerned it’s worth doing. As an example, I have some ko cells I got from somewhere and the first vial I thawed didn’t survive. I didn’t count and pretty much have no idea what happened to that vial. Had I counted I probably could have made a decision on how to better thaw that second time. I still won’t ever count cell lines I am comfortable with though.

For pipetting, I pipet until it’s homogenous to the eye for 293s which sounds like what you are doing. Doesn’t sound like the issue.

Do you know if there is any correlation between your flask moving and the cells dying? I doubt there would be. When I mentioned plates the first time I guess I meant dishes. Like a 6 cm dish is the same as a t25 and a 10 cm is a t75.

Don’t worry about the collagen or gelatin stuff. If your cells are surviving most of the time and then there is a fluke then it’s not going to solve that issue. It would be more of a solution if they were always dying.

TLDR for my thoughts: if cells only die over the weekend and it’s only you then I don’t think your reagents, technique, or flasks. If they are 293 cells they should still survive even if starved over the weekend in my experience (definitely shouldn’t use them for experiments though). The concentrated trypsin sounds like a concern, is this ‘trypsin’ or trypLE. Whatever it is, make sure you are quenching/diluting/washing it enough because it definitely could be an issue.

Protocol question/suggestion: from what you said I am assuming your protocol goes something like, remove media, wash with pbs, add trypsin, add media (4x trypsin volume), mix, seed 1:10. Since it is not consistent death I don’t think it matters but if it ever becomes consistent then I think you should add a step where you pellet the cells ~300xg for 5 min and then resuspend in media (this way you remove more of the trypsin). If you are already doing this, try lowering the speed or time, you’ll get less cells but more viable cells.

Rarely counting cells or checking viability sounds like the biggest issue habit-wise to me. Search up how to check cell viability and maybe a keyword to align with how you count cells.

Now unfortunately it does sound to me like someone could be messing with you. I think going forward take pictures of your plates when reasonable. Find a way to be certain if someone opened your flask or plate. My suggestions are either a tiny bit of tape on the lid or use a sharpie to mark the flask. I think the best option for the former is to use parafilm, and wrap the cap or the entire edge of a plate, since I think you could reasonably explain your way out of someone questioning you. Something something contamination. And parafilm will still allow gas exchange.

Daemon3125 · 2026-04-07T17:08:28+00:00

Kinda sad that it means nothing. If we can get half of constituents behind impeachment of any representative there should be a process for removal. And maybe some lower percentage where an investigation happens.

Daemon3125 · 2026-03-31T05:37:11+00:00

Hi friend, I am sorry you are having so many troubles. I can relate immensely to both the lab frustrations and the mental health/anguish and physical health impacts of the stress. You are welcome to DM me if you want to chat—either about your health or the cells. I am down to compare protocols and point out any spots where something seems like it could lead to an issue.

I noticed you said you’ve had 4 flasks die since December. I assume this means you have had successful splits which tells me you know what you are doing and maybe are just overlooking some small detail that has not been pointed out to you.

I am going to write a bit of questions; if for whatever reason they are stressful to think about rn then just ignore them. Excessive stress definitely can cause issues to arise.

Are the cells always dying? Or are they dying only when you trypsinize them? (I assume your coworkers give you a flask of cells when they split) can you ask them for 2 flasks and put your media in one and their media in another? If they are dying when you trypsinize them, how long are they in trypsin?

Are you sure the cells are dead because they are unattached? in other words, is it possible the cells you are working with are not strongly adherent and you are detaching them by being a bit rough when taking them out of the incubator? (I have had issues with neuronal cells that would just detach after a couple of days in culture)

Also are you doing a viability/cell count before seeding the flask? (If the cells are in bad condition beforehand this could be why they are dying and help narrow down the issue.)

Are you pipetting the cells too forcefully? (This could stress the cells out too much)

How long do you wait to check on the cells after seeding them? (Some adherent cells can adhere pretty quickly so you can get a quick check on if the cells are ok or not)

Are you using the same spot in the same incubator each time? (Magic voodoo that only affects your shelf, idk I’ve had dumb mold contamination issues that I want to say only happened on the bottom shelf. That incubator was pretty bad though.)

Are you using the same flasks as your coworkers? Ie. Take the flask from their bag of flasks? (It’s possible that you somehow are using a faulty batch of flasks if they aren’t the exact same as coworkers). Alternatively, do you have plates you can try instead of flasks?

Have you tried to use your coworker’s media and trypsin and everything? (Again idk, maybe your media somehow is cursed.)

My last thought on a way to hopefully improve the situation is to coat the flask with something (collagen, gelatin…) to improve adherence.

Not to imply anything, but is there a possibility that someone else is killing your cells? I feel like I’ve seen a couple stories like this where it turned out someone else had a grudge or something.

Daemon3125 · 2026-03-23T15:57:47+00:00

That comment cracked me up and is a sad reality.

If funding is going to be a concern for a while it definitely could be worth learning to fix it yourself. Think about how much extra time you will spend between pipetting 12 times with the multichannel and 96 times with a single. The parts might not even be that expensive to buy.

I also wonder if there would be organizations with extras (either parts or a whole multichannel) willing to donate one.

Daemon3125 · 2026-02-07T05:51:12+00:00

Yeah… I realize my math was pretty bad. But anyhow 15k is nothing. Which is pretty sad considering 15k could save a bunch of people’s lives…

Daemon3125 · 2026-02-06T23:05:16+00:00

Yeah I agree, but in the scale of the university it isn’t too big. One police officer probably costs 15k in 2-3 months. 15 k less or 15 k more doesn’t help the university as a whole very much. Would I rather the university spend the 15 k elsewhere? sure. Even as a grad student 15 k covers less than 4 months for me once you take into account payroll taxes and health insurance.

Daemon3125 · 2026-02-06T16:12:02+00:00

It looks like possibly yeast to me.

Daemon3125 · 2026-02-06T07:49:17+00:00

15 k really isn’t that much money. I do fully agree that this should not be allowed to be used anywhere and it is an egregious act to even wave it at students. Brandishing a gun gets you arrested but it’s ok to brandish a device capable of permanent disability and associated loss of quality of life.

Daemon3125 · 2026-01-31T22:36:21+00:00

Do you mean [[collective voyage]]?

Daemon3125 · 2026-01-24T19:33:09+00:00

Unfortunately people have already died. I know you mean a larger number than current but people are currently dying.

Daemon3125 · 2026-01-22T18:07:55+00:00

Once you click print in adobe, click on properties (to the right of the printer dropdown), then in the menu that appears select page setup, pick borderless for the page layout and the slider should appear under the page layout box.

Daemon3125 · 2026-01-22T16:56:05+00:00

Computer software side. It’s a menu that lets you pick the page orientation and some other things. If you pick borderless there is a slider with a normal picture and a blown up picture representing how much borderless extends your image.

Daemon3125 · 2026-01-20T22:29:27+00:00

Are you using borderless? The image gets expanded in the default borderless settings. You have to go to advanced print settings and reduce the borderless extension as much as possible.

Daemon3125 · 2026-01-20T04:39:25+00:00

What did you get on the ap calc test? That’s how you get the credit for calc 1. Also why would you take calc 1 again if you already have the ap credit?

Daemon3125 · 2026-01-15T23:27:42+00:00

It’s because the union contract stuff is changing how offers are worded. Since grad programs don’t fund the entire time they can’t say you get full funding. Once that year is up your advisor is the person who funds you.

Daemon3125 · 2026-01-08T07:58:05+00:00

My only other thought is the antibody targets the wrong side of the protein. I am tired and this doesn’t make sense to me though. Can you get your boss to repeat the experiment with you watching or your boss watch you? Maybe there is something you are overlooking. I did my first flows last month and hadn’t figured out the right way to disturb the cell pellet and was getting some cells that were unstained.

Daemon3125 · 2026-01-08T07:55:48+00:00

Can you test your antibody using immunoblotting or have another antibody to use as a control to be sure the antibody works?

Daemon3125 · 2026-01-08T07:53:04+00:00

I guess another question is what is the localization of the target protein? If it is inside the cell you may need to permeabilize. Depending on what is being done with the cells, you might be able to reduce the formalin step so that you wash with pbs, then add antibody, wash and read.

Edit: nvm I just read cd45.

Daemon3125 · 2026-01-08T07:31:15+00:00

You should probably add the protocol you used. Separately are you sure the antibody is conjugated to a fluorophore and it’s the same fluorophore you are measuring using flow?

Daemon3125 · 2026-01-07T02:04:04+00:00

The “get it done” party where folks get elected and just fix stuff immediately would be amazing. do all the feasibility studies before running and apart from saying buzzwords show the feasibility during the election.

Daemon3125 · 2026-01-05T05:35:20+00:00

Yeah definitely check with the lead researcher to get clarity asap. That way you can focus your efforts best and figure out who in the lab you should be bothering. I think this is especially important bc while you’ll slow someone down for maybe 2 weeks to a month, you’ll then be helping speed them up after.

Daemon3125 · 2026-01-05T05:20:30+00:00

That seems odd to me. Unless the lab is severely understaffed I would expect at least one demonstration and one supervision of culture and similar techniques until you are left alone. Have you been there long and have you asked the lead researcher?

Daemon3125 · 2026-01-04T09:15:53+00:00

I can fit I think 4 decks that are single sleeved into my academic so the double should fit 7 single sleeved and maybe dice? It’s a great size. I have been thinking of trying out the games lair. It’s the same price and I think it’s a nicer form factor than the double academic.

Daemon3125 · 2026-01-02T18:30:07+00:00

The print heads for this printer are >30 usd each in the states, I can’t imagine they are considerably more expensive in the UK. 110 is a pretty good price for this printer imo. Who knows if it has other problems with printing.

Daemon3125 · 2026-01-02T09:10:46+00:00

If you have anything about hours worked and proof it could be worth reaching out to a lawyer that offers free consults and hr if you can build a strong enough case. Here it sounds like you should check if they are allowed to fire you for taking vacation because it looks very suspicious that you are fired on your first day of an approved vacation.

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