I am trying to isolate single cells from mouse lung with B16F10 metastatic tumours. Typically, after enzymatic digestion, I filter the cells through 70um filter, followed by 40% Percoll to remove dead cells. Finally, I lyse RBCs using ACK lysis buffer and proceed to staining.
However, as soon as I begin to stain (surface and intracellular markers), the cells begin to clump heavily and I don't seem to get enough cells for flow cytometry in the end.
I am unable to identify the reason for the clumping. Is it because of the tumour cells sticking around or because of too many dead cells? - I see about 40% dead cells after trypan blue staining, even after the 40% percoll step (~3ml percoll for 10min)
Also, I want to know if
1) Using a 40/80 Percoll gradient to separate tumour cells would help. I don't want to pool the lungs from different mice together, therefore is it advisable to layer the cells from one mouse only? I'm scared of loosing too many cells during this step.
2) Mostly I can get a maximum of 3 X 10^6 single cells from one lung. Is that what is usually seen or do people get more cells?
3) Is it advisable to pass the cells through a 30um filter instead of 70um? This is assuming that the lymphocytes (my pop of interest) can pass through it while tumour cells can't.
Any help would be greatly appreciated. Thank you !
1. Possibly the cells are dying and evoking blebbishield emergency program
https://www.researchgate.net/publication/233742776_Blebbishields_the_Emergency_Program_for_Cancer_Stem_Cells_Sphere_Formation_and_Tumorigenesis_after_Apoptosis
2. Possibly the immune cells in lungs get activated to trigger clumping
3. Eliminate Calcium and magnesium from buffers including PBS to reduce clumping and use low speed centrifugations without brake to prevent cell activation.
Good Luck.
Article Blebbishields, the Emergency Program for Cancer Stem Cells: ...
Hi,
Thanks for your response. Yes, mostly likely the cells are dying, which is causing the clumping, but I'm not very sure how to deal with that.
Yes, I am using buffers free of Ca/Mg ions currently.
I specialize in Kidney, heart, spleen cell isolation and from my experience with cancer cells especially from the organs, DNAase is important to get rid of the DNA material to give you a low viscosity solution. There are products you can try that will dissociate your cells (clamps) into single cells. For example, Accumax. Feel free to read about it and can also request a free sample from the company. Remember to check your cells via flow to make sure accumax does not disrupt the cell surface receptors. Have used it in my cells and it works great.
http://www.accutase.com/accumax.html
I am currently isolating tumour cells from the lunges of patients with non-small cell lung cancer using bronchoscopy. I subject the cells to flow cytometry to evaluate their apoptosis with different treatments. I do not have such a problem; however, I do use DNAse and Collagenase D to digest the cells and then pass them through a 70 um filter.
You do not necessarily need to eliminate the dead cells, instead you can use propidium iodide to determine the dead and the live cells in your analysis later.
You can also try using EDTA or avoiding media that contains calcium as this may increase clumping.
You should add DNAse 1 to the digestion medium and remove Ca2+/Mg2+ from the washing buffers, like use PBS or HBSS without these ions (during diggestion the solution should contain Ca and Mg). May be it is reasonable to use EDTA-containing buffer during at least one wash, but I haven`t tried it.
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