I'm growing mammospheres isolated from breast cancer patient and I want to Identify the the markers on the surface of breast cancer stem cells inside the sample. how many cells are needed and what is the procedure for such experiment?
You can find general protocol for flow on BD website or miltenyi, with small variation ad some titration for some Ab needed. number of cells could vary depending of the number or markers you are going to check at the same time on each cells (more markers, more cells); also the smaller is the population you think to find, the larger has to be the number of events (cells acquired at facs). in general, for flow a good number should not be below 10E5 cells/sample, to give you some reliable data. it also depend if you need to fix or permeabilize cells (if not a surface staining),you should increase. very important is have a nice single cell suspension (not trivial sometimes with cancer stem cells or stem cells growing as spheres). then you have to exclude dead cells (use dapi, PI 7AAD, Acqua live dead depending on the instrument and the color you have in your ab panel) and doublets (especially if you are looking for rare events) using a gating strategy using FSC-A vs FSC-H (and or SSC) single cell gating. so look at your marker of interest only inside the live/single cell population, good luck!
Many thanks Dr. Michela,
I'm looking for 2 markers in a triple negative breast CSCs. What do you think the time needed between trypsinization of mammospheres and carrying out the flow cytometry? (the tumor is very aggressive and the mammospheres are noticed to be formed in 24 hours).
I would use:
Unstained control- 10E3 cells
Ab1 single stained (or isotype control)- 10E3 cells
Ab2 single stained (or isotype control)- 10E3 cells
7AAD single stained - 10E3 cells
Ab1+Ab2, Ab1+7AAD, Ab2+7AAD - 10E3 cells each
Sample with Ab1+Ab2+7AAD- 5E5 to 2E6 cells
Ab staining- 30 to 60 min.
Run- 20 to 60 min.
HI Abdulaziz,
What markers are you looking for, they may be damaged by tripsin, so another more gentle dissociation method such as accutase or EDTA may be better!
I recommend a minimum of 100,000 cells is every tube you want to run on the cytometer. Any less than that will not account for the dead volume in the system and you most likely would not get more than one chance to run them as you adjust settings!!
Kind regards,
Steve
It really depends on the frequency of CSC subpopulations in the bulk of tumor cells. As Steve said, if you expect a 0.5-5% CSCs (in adherent cells), a minimum of 100,000 cells to begin with is reasonable. Besides you want to factor in the potential loss of cells during staining process.
On the other hand, it is hard to expect a big number of cells per mammospheres. How big is your mammospheres? If a mammospheres consists of 1,000 cells, you will need 100 spheres for a total of 100,000 cells.
The other tech challenge I ran into is cell dissociation in spheres. You certainly don't want to trypsinize spheres since the enzyme chops away many surface markers commonly used for triple negative breast cancers, e.g. CD44, CD24, CD326. But efficiency of cell dissociation and viability from spheres using non-enzymatic buffer (EDTA) are not good, at least in my hand.
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