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16

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, ...


11

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 ...


11

This is an excellent question, I have been training people to culture cells for about 12 years and students have a hard time grasping this and appreciating the importance etc. What cells are usually experiencing during pipetting is analogous to a crowd of people trying to fit through the doorway of a building. Shearing during pipetting is certainly a ...


10

The storage itself can be done both in the liquid and also in the vapor phase in the liquid nitrogen. Both have their advantages and disadvantages. In my experience there are two main reasons why storage in the vapor phase (although there may be temperature fluctuations when the nitrogen gets low) is recommended. First, you avoid cross contaminations of ...


9

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 ...


9

Clones are genetically identical daughter cells from one parent cell. So when you start you culture from one cell, all new cells are clones of this cell. The first three cells (red, green and blue) undergo clonal expansion. If you multiply cells in cell culture you usually start with thousands of cells, as this can be seen in the second part of the ...


9

The cells never died in the sense that they kept replicating, individual cells still died. They were safely cultured in petri dishes before Henrietta Lacks died. The cells came from a tumor that developed from her cervix. The cervical cancer cells had developed high telomerase activity. Telomerase builds telomeres on the ends of DNA, protecting the ...


7

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.


6

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 ...


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 ...


6

Besides the etymologic explanation that @aandreev gave, in cell culture this term is commonly used to describe the density of adherent cells and it is used as a measure of their proliferation. It is usually combined with an estimated (or counted) percentage, so 10% confluency means that 10% of the surface the dish or flask used is covered with cells, 100% ...


6

TGF-beta would be a good candidate. To cite: "TGF-β inhibits G1/S progression in a variety of eukaryotic cell types. Among these, untransformed epithelial cells are particularly sensitive to the growth inhibition by TGF-β." http://genesdev.cshlp.org/content/14/24/3093.full Fetal bovine serum (FBS) contains a high level of latent TGF-β. Human serum as well ...


5

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 ...


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

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 ...


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 ...


5

I think HELA cells are edible, although from moral point of view this would be cannibalism. Despite they are cancer cells, they are safe for foreign organism, because any ate matter is destroyed. Even if these cells were implanted into another being by surgery, they will be safe since immunity will recognize them as foreign and kill. They are much less ...


5

I found a protocol by ATCC for NTERM2 cells, and it didn't mention any specific flask, so any cell culture flask would do. Since ATCC is basically a cell culture bank I trust that their protocol is valid.


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

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

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 ...


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 ...


4

Great idea! It seems that this research has been done before and your hypothesis is correct, but testing a wide variety of alcohols and testing your hypothesis for each one of them would be a great project. Now, to do this, I would expect you would need to check the growth of the cells after certain periods of time. How would you do this? Well, the best ...


4

Based on T Abraham's answer, a hemocytometer would work. However, a hemocytometer requires a microscope, but if you are in a cell lab, you probably have access to a microscope. I would recommend using mammalian cells instead of bacteria, they're larger and easier to see, and more relevant to human health. If you're interested in liver toxicity in particular, ...


4

One is not normally required to serially dilute E. coli cultures for spectrophotometric measurements, at least in the experiments where the OD value is important. For most protein expression work, the consensus is to start the induction at OD 0.6, and for most chemically competent cells, the optimal OD ranges from 0.3 to 0.8 depending on the competency ...


4

It's because E.coli BL21(DE3) are competent cells. The competent is the key here as the cells were chemically treated so the transfection can be performed by heat-shock with high efficiency. This means these bacteria are quite fragile, due to the chemical treatment, and therefore are very sensitive to both mechanical and thermal shocks. Pre-heating the ...


4

It is a well documented observation that Plasmodium (vivax and knowlesi) infection is dependent on the Duffy blood groups [1]. Individuals lacking the Duffy antigens (Fya and Fyb) have lower susceptibility to malaria. Plasmodium expressed Duffy Binding Proteins facilitate in establishing the initial contact between the merozoite and the RBCs. However, ...


4

Bacteria or other microorganisms cannot really grow on anhydrous (totally dry) glucose because they need water. However, they can remain there and cause contamination. Even if you haven't actually touched the glucose sample, there are many bacteria suspended in the air and they may settle down in your glucose packet. When you take the glucose out for ...


3

I realize this is an older topic, but I'd like to clarify some misconceptions: 1) DAPI is usually referred to as a semi membrane-permanent dye because its penetration through viable cell membranes is concentration dependant. At lower concentrations it is mostly excluded from viable cells, while at higher concentrations it stains live and dead cells ...



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