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fraffly

Member Since 06 Jun 2012
Offline Last Active Dec 12 2012 06:25 AM
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Posts I've Made

In Topic: Is this contamination or cell death?

12 December 2012 - 06:20 AM

Doesn't syncytia occur quite commonly in some cell lines ? I've observed similar looking cells multiple times in different cell lines in different labs. I think some retroviruses can cause that too.

There seem to be quite a few multinucleate cells (if they are syncytia) around in those pictures though, might have something to do with the selection pressure due to passaging behaviour. In my experience they adhere much stronger to the plastic than the "normal" cells, so they tend to accumulate when the same flask is used for several passages.
Changing flasks more often helps to reduce their number.
Also, since the normal cells usually detach faster than the syncytia, it's easy to time the trypsinization and just passage the normal cells.

In Topic: Immunofluorescence on cells - can I keep the coverslips unstained in PFA?

12 December 2012 - 05:47 AM

I'd store them in PBS. We usually just pipetted the PFA off after fixation and added PBS, then there is also a rest of PFA present. This way the cells stay quite stable for a week or two, we never checked longer than that.
Don't forget to quench free aldehydes before labeling, they can bind to the primary Antibody and give false positives, we used
50mM Ammonium chloride in PBS for 5-10min on 4°C.

In Topic: Performing MTT assay in suspension cells (new in the subject)

12 December 2012 - 05:07 AM

If you culture the cells in suspension, keep it that way for the assay. Centrifugation doesn't bother these cells much.
For suspension cells, I'd incubate in round bottom well-plates, then you'll get a nice pellet when you centrifuge them. In flat plates no compact pellet can be achieved and you might loose some cells by pipetting the medium off.

- centrifuge (for PBMCs 300g/5min works well)
- remove medium
(opt. make a washing step)
- add MTT solution, resuspend cells, incubate
- dissolve crystals, I've used DMSO, it works fine. If you're in doubt, just test the different solutions.

In Topic: How to avoid crystal formation after fixation by formaldehyde

31 July 2012 - 02:32 AM

Just out of curiosity, why do you dry them ? And what are you doing with them afterwards ?
I've done immunofluorescence for conventional & ElectronMicroscopy. What we usally did was, dip the slips shortly in dest.water to get rid of the salts and if you need them dry, hold a corner of the slip on a tissue to suck off the water.
If they're used for immunofluorescence afterwards I'd recommend to use a quenching solution (e.g. 50mM Ammoniumchloride in PBS) after the fixation to reduce background and false positives from free aldehyde groups, which can bind to your antibodies.

In Topic: Dilution problem

25 July 2012 - 02:30 AM

Your concentration is too low, you can't dilute 424ng/ul to 500ng/ul, you'd have to concentrate it, for example by evaporation.
But in my experience, when sending samples for sequencing they always want a concentration much higher than the one they actually need for a successful sequencing, they like to play it save. I've regularly used concentrations 5 times lower than demanded and it always worked well.
I'd send it in the concetration you have, it'll work.
Plus, measurement of DNA is rarely precise anyway so you're in a good range.

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