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This Bitesize Bio article is very informative on this issue. The 1970 study they cite found negligible reduction in the the efficacy of non-beta-lactam antibiotics (kanamycin, chloramphenicol) over spans of 4 weeks or 60 days. Ampicillin loses about 10% activity at 4 weeks. In my lab, we only take care not to use LB/Amp plates that are older then 4 weeks, ...

I find that I only get clumps (with HeLa and similar cell types) if I leave them in Tryple too long. Generally I leave them in room-temp (not hot) Tryple for ~8 minutes, then I angle the dish, lid off, so that I can see the "sheen" of cells on the dish (this happens in the hood, of course). Then I blast the sheen with Tryple using a 1000ul pipetter, which ...

Try perhaps lowering the level of expression of your construct. HEK cells are notorious for expressing large quantities of your transfected insert. Do you have it under a CMV promoter? Try a milder one, such as an ubiquitin promoter or similar or, even better, a doxycyclin-inducible system (e.g., clontech's tet off) so you can manage the expression levels ...

AbCam suggests HeLa cells as positive controls for their antibody to GLP1R. They provide the following pictures of HeLa cells labeled with their antibody: (The image of the right is treated with synthesized peptide.) According to Wikipedia, GLP1R is also expressed in pancreatic beta cells and the brain.

Assuming that you are talking about E. coli: As long as you are resuspending the cells in a suitable liquid, e.g. fresh medium or buffer, then from my experience you don't have much to worry about - the cells are very robust. I've found that different strains and growth conditions give pellets with very different qualities - some will resuspend quite well ...

First thing I'd do is replace the HEPA filter. A copper plate/foil may help, and it certainly won't disrupt the flow of heat, as copper is incredibly conductive (that's why they make electrical wires out of it), and poking holes would help with the airflow, but a new filter will probably make the most difference. I've never used a full-copper incubator, and ...

It's an easy experiment to do. Take your cells aliquot them into 10 microfuge tubes, and pipette each suspension increasing amount of times, stain with trypan blue and count. The most important factors will be which pipette-type you use; I would expect a p1000 to cause more damage then a p200 then a p20 due to velocity of the fluid. Also the most important ...

Cell clumps that not dissociate with rigorous pipetting might very well be caused from free DNA in your cell suspension (i.e. if you trypsinized for too long). Free DNA attracts cells which bind altogether forming clumps. Two possible ways to get rid of these clumps: Centrifuge your cell suspension at 5000 rpm for 2 minutes with acceleration on (at 4) and ...

In some protocols for setting up primary cultures (for example from mouse bone marrow or rat endometrium), there is a step that requires pushing cell suspension through a large needle to get rid of clumps. Of course, this carries a high risk of damaging the cells, so sometimes collagenase is used instead. It might also be helpful to wash your cells with PBS ...

I recently tested exendin on INS1e cells in an Edu incorporation assay (similar to BrdU incorporation) to observe if this compound induces proliferation of the cells. Compound incubation was for 24 hours. I saw no incorporation of EdU with this compound over untreated levels. In addition, during the assay I look at overall cell number with a DNA stain, ...

I've worked with MIN6 cells in the past, and have found that they can take at least 12-24 hours to fully seed (if they are being cultured on polystyrine). Depending on the specifics of your experiment, and whether doing so would complicate your controls, you might also want to try coating the surface you are attempting to seed on with laminin. MIN6 cells ...

Searching literature to see what the published protocols were is the best bet. I found a simple document for INS-1 cells here. There is also this paper describing the isolation of an Ins-1-derived cell line 832/13 which my lab (though not me personally) have experience with. Cells should only be sub-cultured when nearly or fully confluent. With Ins-1 cells ...

2-me is a reducing agent necessary to be added to help keep free radical oxygen from affecting mouse cells. It is generally not necessary for human cells. Also from S Bannai and Ishii et al., 2-mercaptoethanol improves tumor cell uptake of cystine by creating a reducing environment.

If you are you working with primary cell cultures or cell lines may be you could do this when cells are in suspension, after tripsinization, mix them well with a 5mL serologial pipete in a falcon 50mL tube and plate to a 100mm2 with a final volume of 7mL. And finaly draw an eight with the plate in the hood more than 3 times to mix them well. Edit: For the ...

Senescence does not equal cell death, but is simply an arrested state in which the cell still remains viable (1). It has been shown throughout the literature that this state corresponds with an altered cell cycle profile different from contact inhibition or damage-induced arrest (2). This corresponds with an enlarged cell size, altered gene expression (3, ...

DAPI and Hoechst 33342 (there are different Hoechst dyes, 33342 is one of the most commonly used) have very similar spectral characteristics. The only point is that DAPI is much better excited at 405 nm than Hoechst. Some microscopes and flow cytometers nowadays have 405 nm lasers or LEDs instead of UV sources, so this can become relevant. The main ...

The Hoechst 33342 dye is similar to DAPI in that both are UV-excited, minor groove-binding, and emit signals proportional to total DNA content. Both are maximally excited around 355 nm and emit around 460 nm. A UV light source is required, which may harm the cell. However, this is only a risk with Hoechst, as DAPI requires the cells to be fixed and/or ...