What are good cell cycle markers?

Dear all,

I have some cells that I want to stain for Annexin V, but they were already fixed by PFA. Can I still do immunofluorescence on Annexin V?

Also, do I need any special buffer? As for living cells, it needs Calcium to work.

Thank you!

I don't understand the concept behind FSC-A vs. FSC-H for getting singlets from a FACS data. Can someone please explain it to me?

Hey all my cell culture fellows,

i need your help and experience...

i am trying to establish a cell line in our lab. i bought a fish cell line and i am now trying to get it startet. But it is not going as well as i hoped. The epithelial like cells are growing at 19°C with no extra CO2. I got about 1 Million cells delivered and i tried to seed them into a 25cm^2 flask, as statet in the protokoll. at first they seemed to grow fine, but in the last passage and i the passage now, the cells are distributed very unevenly in the flask. Last passage, the cells were very dense in the upper half of the flask (like if you watch the field under the mikroskop), and in the lower half, they were not as dense. And around the edges there were almost no cells growing.

In this passage, i have a small field in the middle of the flask where the cells are very dense, and around that they are not growing well. Also today on day five i have a lot of cells detached. Its very frustrating... i thought maybe the incubator is not standing right, but the distribution of the cells make no sense to me...

Has anyone any ideas?

Thank you!

I try to find an equation for optimizing voltage and time in transferring proteins.

Hello All,

Please i am looking for someone to explain how to seed 10000cells per well in a 96 well plate. After cell counting, I have a cell viable conc of 3.348 X 10^6. I just want to be sure aIm doing it right before going ahead to run my plate-based assay. I am trying to run a cytotoxicity test using a ready-to-use MTT reagent another worry is which seeding density is best 10000 or 20000. I intend to seed and differentiate the same day for 3 days before running the analysis. Thank you

Hi everyone! I have stained embryos with JC1 and now I am trying to quantify with the ImageJ software. I got values like area, mean, mode, integrated density and raw integrated density. My question is which value (integrated or raw integrated density) should I consider for quantification? And after considering any of these parameters (integrated or raw integrated density), do I need to measure corrected total cell fluorescence or I can directly use it as fluorescence intensity? Thanks in advance.

Hello,

I am using Prism to calculate the IC50 of different chemotherapeutics. This time, my concentrations are between 0.25 and 1.5 microgram/mL (0.25, 0.5, 0.75, 1, 1.25, 1.5).

Converting the concentrations into log gives me problems this time since 1 is one of the concentrations used and I would usually put 1 as my non-treated control (log1=0).

How would you put the concentrations into Prism? convert it into nanograms/mL and then use 1 as the control point? I am wondering if that would change the value of my IC50 because the curve gets longer the bigger numbers I put in, in comparison with my control.

Thanks!

I want to quantify the fluorescence image of the cells by using image j software but I am confused between the mean gray value and integrated density value and its appropriate use.

Hello there.

After adding the protease inhibitor cocktail to the ripa solution, I aliquoted it at room temperature and kept at -20 degrees.

However, my aliquot process at room temperature took about 15 minutes and I did not do this on ice.

Would I damage the RIPA solution under these circumstances?

Thank all.