Hello,
I am isolation cancer stem cells from MCF-7 cells. I saw protocols for 96 well plate where 1 cell per well is seeded. How many cells should be seeded in 6 well plate.
Hi Heena,
Beneath please find a protocol for cultures using 6 well plate you required. Upon receipt of cultures, the following describes
procedures for plating a single cell suspensions in 6 well plates, for
Tumorsphere formation.
Other Equipment and Reagent Requirements
From a T25 Flask (1 x 106 cells)
1. Transfer the cell contents from the T25 flask into a sterile 50
mL conical tube.
2. Centrifuge the 50 mL conical tube at 200 x g at 4℃ for 15
minutes.
3. Carefully remove all but approximately 100µL of the
supernatant using a pipette.
4. Gently resuspend the cell pellet in 12mL of fresh Cancer Stem
Premium™ medium (catalog no. 20101-100, or 20141-500),
pre-warmed to 37℃.
5. Aliquot 2mL of the cell suspension into each well of a single 6-
well plate (VWR, Cat. No. 29443-030).
6. Transfer the plate to a 37℃/5%CO2 cell culture incubator.
7. Add additional fresh Cancer Stem Premium™ medium at a
volume equivalent to 10% of the total culture volume every
two days to maintain the culture over a period of 7-10 days.
8. Tumor spheres will start to a form, and will be visible in
approximately 2-3 days, and after day 2 cells may be
passaged or used for experimentation.
Best wishes,
Ilya
Hello
yes this good protocol .
And see links and pdf .
Good Luck
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708191/
http://www.promocell.com/fileadmin/promocell/PDF/Cancer_Stem_Cell_Medium.pdf
Thank you all,
I have isolated Cancer stem cells but there are number of cells which are present in tumerosphere. How to characterize that the are stem cells ? Most of times FACS is the viable option. Are there some other methods we can use like PCR of some markers or Western blotting etc. Please suggest.
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