I recently checked and found that my KHYG-1 cell line shows 2 positive peaks for an intracellular protein that I am trying to study. I would expect any cell line to be homogeneous in this sense. Does this indicate contamination? What other factors could lead to this? Thanks.
In general, the individual cells in a cell line can be transformed and gain their own phenotype which depends on the number of passages. You can not take cell lines granted. However, the type of assay your are doing also can give you heterogeneous populations sometimes. If you are using intracellular staining, part of your cells in the antibody staining might not be uniform. I would suggest you to check for both the protocol (and more repeats of the staining) and the cells using different techniques such as microscopy. I hope this helps.
Hi Riya. Did you run isotype control or FMO for gating positive cells? In case you used FMO, try running isotype control and see whether first positive comes in that also? Sometimes staining also increases autofluoresce of cells resulting in a shift in fluorescence. Titration of antibody can also help if you expect cells to be homogenous for expression of your target protein.
Good luck
Dear Ravi,
Thanks for your suggestions. Even if the first peak is actually negative (which we will know after using isotype control), isn't it still a problem that my cell line isn't homogenous? I am having trouble figuring out if this is acceptable? In my opinion, working with cell lines having heterogeneous expression is an issue. Thanks!
Dear Ramachandramouli,
Thanks for your response. We have repeated the staining several times, pretty sure the protocol is okay since it works well for other primary cells we are working with for the same protein. Also, I wonder whether microscopy will be able to pick up the differential expression in this case? Is there any way to check the cell lines for authenticity? I understand the cell lines become transformed with passage numbers etc, but what I am curious to know is whether this is an issue that needs to be sorted out before I start my experiments? Thanks!
Dear Riya. If your first peak happens to be negative, then you need to rule out insufficiency of antibody. If the results remain same after titration of antibody, then only you can say that there is heterogeneity. I mean, we have to be experimentally correct before reaching on a conclusion.
Good luck
Dear Riva,
It depends on the kind of study you would like to go on the cell line, before proceeding for your experiments. However, if the same cell line is available with other labs or if you have any old frozen stocks of the same cell line, I would suggest you to check them first for the same staining and compare the percentage of low expressing and high expressing cells. I hope the cells are looking uniform (morphology) under light microscope. This is also an important observation to make sure. Sometimes, cell grow in groups or clusters, where the cells inside the clusters behave differently compared to the outer cells, which have differences in cell-cell contacts. And also, perform an experiment where you expect a change in your protein expression and compare the peaks again. If a flow cytometer differentiating the peaks means, you can see differences in confocal microscopy, which will also give more information about precise sub-cellular localization of the proteins which might be more important for nuclear proteins or vesicular proteins. Sometimes, cell size and cell clumps (doublets) also matters for flow cytometric analysis. Use EDTA in your buffers if it the latter case. I hope it helps. Goodluck
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