Ht-29 transfection with pCMV6-X?

Hi Diana,

I am afrais that you will have to check the efficiency for your cells in your conditions. I have transfected HT29 cells from two different labs (over time tumor cell line cultures gain variability) with very different results. 10-20% efficiency in one case, 70-80% in the other. With a little of luck you might get the second.

I suggest you tried transfecting with a EGFP vector (similar size and promotor to pCMV6) and quantify transfected cells by fluorescence microscopy or flow cytometry.

DNA quality is very important also.

Goog luck

Diana Cadena

Thank you very much for your quick answer.

I have another question please. I found several trouble with trypsination. It works but it takes a lot of time and I have a lot of death.

Furthermore, for flow cytometry because I heard that I should use EDTA and avoid trypsine. I tried EDTA, but, I failed. Thus, have you got some advice for that.

Thanks in advance

Best Regards

Diana

Konstantinos Stamatakis

Hi again,

There should not be a problem with tryspinization. I use 0.05% trypsin in EDTA (0.016% EDTA in PBS) and they come off fine, in 5 minutes. I wash with PBS (Ca and Mg free) before. It is not good to leave them too much time in trypsin-EDTA (more than 10-15 min), that could increase cell death. Also, it is recomended not to tap the flask in order to make the cells come off, it causes them to aggregate. In any case, if you have problems with trypsinization, try washing first with PBS, then wash with a little bit of trypsin solution, remove it imediatelly and then add new trypsin solution and place them in the incubator for five minutes. After that, check if they came off the plate and if that is the case, add at least double volume of medium with serum to kill trypsin before centrifuging. This is very important (I am sure you already know this) because the cells are very sticky at this point and you need proteins in solution to keep them apart. If further washes and centrifugations are done, you can add 0.5-1% BSA in PBS to do this (except for fixation).

I have used HT29 for flow cytometry, trypsinizing. There was no problem. I get less than 5% of apoptotic and necrotic cells this way. Long time exposure to EDTA is as bad as trypsin.

Another issue you should check, especially if you have doupts about the cells (although this should be done rutinely), is to check for mycoplasma infection. They alter all cell responses and they can affect growth (not necessarily negatively), trypsinization, response to drugs and transfection efficiency.

Good luck!!

Ted Bremmer

Transfection efficiency is definitely affected by the efficiency of trypsinization. It will also be important for HT-29 to have reagents that are light on the cells and don't lead to excessive cell death (see https://altogen.com/product/ht-29-transfection-reagent-colorectal-adenocarcinoma/). There can be issues with intracellular delivery tied to the size of your plasmid, but those are generally resolvable with complex condensers.

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