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15

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, ...


7

In my opinion wearing gloves during cell culture is a good idea. It works both ways as a protection: For your cells since you are not loosing small danders from your hand and for you since you are not getting media or chemicals used in cell culture on your skin when you are uncautious. I usually prefer nitrile gloves over latex, since nitrile gloves are ...


6

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 ...


5

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.


5

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 ...


5

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 ...


5

The answer is that the majority of the cells were frozen from very early in their Hayflick lifetimes e.g. after 9 population doublings. They have been thawed out judiciously and only as needed thus preserving a lot of frozen stocks. When an ampule of cells frozen at, for instance 9 population doubling, is thawed, the cells pick up where they left off and ...


4

As far as I know, that is fine. Thats the temperature that Amp is regularly stored at. I always do a negative control anyways to ensure that my antibiotics are still lethal. Just don't stick it in the microwave to make plates


4

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 ...


4

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 ...


4

After looking around for a bit, I came across a few potential solutions however they are not a definitive answer to your question and requires testing under your experimental conditions to see which works best. This page contained one excellent suggestion and that is to select for cells that do not clump during the passage, by pooling the cells in 15ml of ...


4

This is a very good question and it highlights a very frequent misconception about cell culture and it is using the passage numbers as an indication of how well a cell is behaving. Although this can be a good barometer but it is misleading. Passaging can stress the cells if performed very frequently, although again that depends on how fast the cells are ...


3

Extracellular matrix (ECM) fluoresces, especially Collagen and Laminin. The maximum is in the DAPI and FITC channels and the fluorescence becomes weaker towards longer wavelengths. However, since the coat on the TC flasks is very thin, I would not expect this to be a problem. The best thing is just to try it. There is also a quite famous document available ...


3

Anecdotally I have not observed any cell death upon pipetting of E. coli DH5alpha or TOP10, however as competent cells, mixing by pipetting up and down is discouraged due to the compromised cell wall.


3

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 ...


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 ...


3

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 ...


3

I don't know about solutions, but we keep LB-Amp/Carb/Gm plates in 4°C for two-three weeks at a time, and the antibiotics seem to work.


3

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 ...


3

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, ...


3

I don't think it's wise to use a spectrophotometer for this experiment, as it is a total count, i.e. it can't distinguish between dead and alive cells very well, and so she might not see a clear difference between her treated and untreated algae. If she has access to a haemocytometer and a good microscope, then she can count the living cells directly, and ...


3

Some of the reasons why immature blast cells are studied: They have self renewal ability Can be differentiated to different types of cells Serve as model for studying development: this is quite pertinent to the gene expression studies. Because it is important to know what changes take place during differentiation and simply looking at the differentiated ...


3

The website you want is The Cell: An Image Library. They also have movies. A quick search of 'Homo sapiens tumor' revealed several Creative Commons videos of tumor cells moving around in vitro with attribution and no annotation or anything else. Sometimes the site can be a bit buggy, but it's a pretty awesome resource when it works.


3

The classic example, though I am sure there are others that are smaller, is the slime mold Dictyostelium discoideum: It can have up to 100,000 cells and exists as both single cells and as a multicellular organism (emphasis mine): Dictyostelium amoebae grow as separate, independent cells but interact to form multicellular structures when challenged by ...


3

This is the original 1972 paper describing the isolation of the S2 cells from Drosophila. However it would be best if you specify what application you are planning to pursue with the cells, which would make it easier to suggest which ones to isolate. I have personally isolated garland cells, which are visible with the naked eye and highly suitable for ...


2

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 ...


2

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

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.


2

We typically use High Glucose as cells grow faster. One caveat is that there are concerns that certain protein modifications (specifically OGlnAc-ylation) may be upregulated in non-physiological (25mM or high glucose) media.


2

My main concern here is why you are using suspension-culture selected cells for an adherent cell-based assay? Is it possible to obtain a clone of adherent HT1080? Depending on what you are assaying with your protein of interest, the cell biology may be altered changing from suspension/adherent culture systems. Here are my experiences using PC12 cells in ...



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