I'm interested in staining cells (breast cancer cell line) with an antibody against a cytoplasmic or nuclear localization, then go to the FACS and sort them out (positive and negative separately). Is there any way of performing a microarray or some similar analysis with these cells, which have to be fixed and permeabilized during the staining protocol? How to be sure about the integrity and quality of the DNA and/or RNA?
Thanks a lot Mathias... I'm not really sure if it's possible to do it... i'll see
It helped me knowing that I'm in trouble :) yep, I've been looking for an extracellular marker without any good results... And yes, I've been told to use RNALater... I wanna be sure, or at least close to with some probability, that it'll work before doing it since it's a lot of work (and money) to expand cells for one week or 10 days and then sort (it's a tiny proportion of the population that i'm chasing). Cheers!
Are they fixed prior to staining with methanol or paraformaldehyde? It is not difficult to obtain mRNA or miRNA from stained cells, the problem might be the degradation. If they are over-fixed this becomes difficult too...have you thought about using laser capture microscopy on a few fresh-frozen samples you've stained? If it is possible because you need just a few and have only a few stained cells? This might work better than FACs...but again it depends upon rarity. There are also instruments that can help with single-cell analysis (one the opposite extreme)...Stem cells can be notoriously difficult to find, stain and extract RNA from, but there are ways around the low abundance difficulties.
here is one manuscript on single-cell analysis....http://www.ncbi.nlm.nih.gov/pubmed/23106823 There are many more...you might also try Nature Protocols...hope this helps!
Hi Jennifer, thanks a lot for the advices. I'm trying to isolate a subpopulation that is around 5% of the total population. I usually use paraformaldehyde for fixing the samples for immunofluorescence and I was thinking about using the same for immunostaining and then sort them through FACS. I have no experience on laser capture microscopy but I'll consider it, thanks!
Completely understand. Still writing the paper now, but I did lots of counting based upon morphology and its painful to find small populations! Another possibility is to use a vector to express your protein of interest in cells that normally transcribe it at higher levels. You can email me with any questions you like and I will do my best to help or ask my friends at different institutions if they have ideas.
Have you considered not fixing your cells prior to staining & FACS? When I did FACS on stem cells I could not fix the cells as I needed to passage them afterwards.
Thanks a lot Jennifer! No, Lisa, I haven't considered it. I've never tried it. Would it be possible to permeablize the cells, stain them and sort them? Thanks
Mariano, (been on vacation) when we did the stem cells, we used 6 different Ab simultaneously, some for internal and some external sites. We didn't have any issues with getting the intracellular Ab into the cells. I have attached our methods we used.
And this is our interpretation of the company's protocol:
I hope this helps.
Hi Lisa, I'm still on vacation :) Thanks a lot, I'll take a look to the protocol and I'll give it a try. Thanks again!
Hey Mariano, did you get any successes. Was hoping to do something similar
Hi Alex, well, I didn't try as we decided not to go for RNA analysis and focus on the protein level. But I don't exclude doing it in the near future. And both approaches suggested by Lisa and Jennifer seem to be promising.
Hi Pia, oh, ok, thanks a lot for the tip, sounds very good... and the cell number should be around 10^5 too. Should give it a try. Thanks!
Dear Haoyu Liu, I have finally decided to analyse these cells through a different approach so I finally didn't tried it. Sorry. I hope it works for you
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