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Help me lab gripes, you're my only hope [May. 24th, 2012|02:11 am]
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lab_gripes

[dune_drd]
So I'm doing long-term fluorescent studies on Hela cells. Well, I used to do them in another lab, ever since we moved nothing is working anymore.

Whenever I now try to image, my cells die. I tried everything; different cell batches, different buffers, gentler transfection methods, but nothing works. Whenever I set up the cells on the microscope, they bleb and die. And I mean all of them. And even if I manage to image some before they die, that's really not the point of long-term measuring, you know? I am completely clueless, and the boss is getting impatient. I have installed a heating stage and use phenol-red RPMI supplemented with Glutamate and 15mM HEPES. I only remove FBS, which I don't want to interfere with my measurments. This has always worked before, a little starvation was always okay, but not anymore... any idea what could cause this? The cells look fine in the flask, but when I transfer them onto glass my problems start. :(
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[User Picture]From: yelya
2012-05-24 01:24 am (UTC)
If you have moved, it means that you are probably using another incubator? Have you checked it? Is it working properly, 5% CO2? Are there other people using this incubator?
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[User Picture]From: dune_drd
2012-05-24 09:23 am (UTC)
The incubator is fine, the cells are fine as long as they are in the incubator... the problems start when I move them onto the microscope, away from the CO2...
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[User Picture]From: vindonnus
2012-05-30 03:48 am (UTC)
Has the incubator been checked with a fyrite recently?
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[User Picture]From: ammonoid
2012-05-24 02:57 am (UTC)
Is it new glass? (what glass is it? coverslips? Incubation slides?) If the glass has been used before it may have stuff adsorbed onto the surface that is messing with your cells. It can be removed by acid cleaning.

Otherwise, I dunno.

I thought HeLa cells were indestructable. Maybe you should patent it when you figure what it is.
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[User Picture]From: dune_drd
2012-05-24 10:10 am (UTC)
I use incubation slides, more specifically Labteks, so at least medium supply should be no issue. I tried etching the slides beforehand, didn't improve the results :( . I'll try again with HF, just to be sure
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[User Picture]From: dravogadro
2012-05-24 04:06 am (UTC)
Can you explain your setup a bit more?

Are you growing them on the glass the night before? It sounds like the glass is not acid-washed properly? Has the laser power been accidentally set at an obscene value?
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[User Picture]From: dune_drd
2012-05-24 10:14 am (UTC)
I use a simple wide field microscope with a heating ring on top - and yes, I checked the laser powers, but it's the same as in the other lab before, when the cells were fine... I actually grow the cells for 3 days now: one they are allowed to adhere, then I transfect them on the second, imaging is on the third. I didn't acid-wash the slides, as I didn't before, but I'll try again...
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[User Picture]From: small_chicken
2012-05-24 07:10 am (UTC)
I've heard that lot numbers of FBS, even within the same company, can vary a lot and really screw around with results.



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[User Picture]From: dune_drd
2012-05-24 10:15 am (UTC)
Our assistant checks the FBS almost religiously, I don't think it's an issue. I asked her several times, but our lot number is still the same as it always was
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[User Picture]From: dr_bob
2012-05-24 09:04 am (UTC)
The cells that say HeLa on the tube could be something different. It took me over a year with one system I was setting up to realise that just because a tube says 293 cells on it, does not mean it will work with the assay. I'd suggest that you try to get hold of a vial of cells from the previous lab and see if you can get it working in that context. The same goes for an aliquot of the FCS batch, the glass/flask you used (which may have a different treatment on the surface), the DNA sample (some qiagen preps are not too clean in my experience) if you have access to older aliquots.

Other possibility is that the laser power is way too high, and you need some sort of filter to reduce its intensity. If Blebbing on the microscope is your outcome, try it without transfecting to eliminate that part of the process.

Also, try 1% FCS rather than none, if that is compatible with your assay.
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[User Picture]From: dune_drd
2012-05-24 10:16 am (UTC)
I thought that as well and made our assistant thaw new cells that she had recently bought from a cell company, so I'm pretty certain it's nothing wrong with the cells themselves. I even acquire some other Helas from another group, but they die as well :(
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[User Picture]From: cheez_ball
2012-05-24 04:16 pm (UTC)
HeLas have specific markers. You can order primers and do a quick PCR to verify.
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[User Picture]From: cheez_ball
2012-05-24 04:19 pm (UTC)
I had this problem when my incubator crapped out on me. Things appeared to work until I tried to actually do my experiment for realz.

Could be a number of things. I would start with trying to do this transfection without the glass. Just use a 24 well plate, 6 well plate or something that should work. See if the cells still bleb out and die.

Are your non-transfected controls also blebbing and dying when you put them on the glass?
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[User Picture]From: chaotropic
2012-05-27 07:17 am (UTC)
im assuming you tried the same exact conditions (on the glass, in the same chamber ect, without the microscope) and have seen your cells are fine? if the glass is PDL treated, it could be that it was not washed well?

otherwise, it sounds like it has to be your microscope, probably the excitation illumination? tried to lower the exposure as much as possible and/or throw in some neutral density filters?
also, its been awhile since ive used laser excitation, but my thought was that the laser power actual numbers might vary from set-up to set-up so even if you are using the same numbers, you might actually be getting different intensity. (also laser output declines with age - and changing microscopes may actually be exposing the sample to brighter excitation intensity since some lenses transmit better than others).

let me know if you figure it out! i do a lot of microscopy and am about to move from Portland, OR to Springfield, MO, so these things worry me...
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[User Picture]From: dune_drd
2012-05-27 03:58 pm (UTC)
Thanks for your suggestion - too much power was actually my first idea what's wrong, alas, I fine-tuned the setup and it's exactly the same on the new setup... I'm sure it's something biological, all the technical stuff I already tested (and at least that can be done reliably *g*).

I get the impression my cells don't do so well with my reset at 4°C (which I do beforehand), but the fridge is fine, etc.etc. I don't get it :/
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