Hello. I have a problem with getting sphere from the AGS cell line. I want to get sphere from this cell line to receive cancer stem cells but its really hard to get sphere through low attachment plate by adding growth factors. Is there any idea or any new method to get these spheres better and easier?
Related references:
1. Zhang, Xiaowei, et al. "Identification of stem-like cells and clinical significance of candidate stem cell markers in gastric cancer." Oncotarget 7.9 2016.
2. 西川晋平. "Aldehyde dehydrogenase high gastric cancer stem cells are resistant to chemotherapy."
Hi,
I don't know what method you follow at the moment. I use plate centrifugation method (V shape bottom plates) and it worked for me with different cell lines including stem cells. Having said that, I didn't use AGS cells before but I found 1ul of 10% PVA per mL media can help in spheroid formation. I did the spinning at 700G for 5 mins. If you don't have access to a plate centrifuge then you can make them separate using eppendorf tubes. You can see the aggregate immediately following the spinning and the spheroids should form within 24 hours, some times it takes 48 hours.
HOPE YOU FIND THESE TIPS HELPFUL.
Good luck
Hi Rasool,
Got your protocol dear..there are some changes you need to make in your protocol.....Make EGF and bFGF at 20ng/ml...use 2% KSR if possible....Make a complete media containing all supplements, try not to use antibiotics .... Don't grow spheroids in T-25, they are not good for spheroid culture....use T-25, seed 20-50,000 cells/well....Add 2 ml media on first day......and another 1ml (complete media) on 3rd day....and 1ml on 5th day..dont change media....You will get spheroids on 7th day...dont culture them till 10th day as it will induce apoptosis with more time..
I hope it works, if don't then last thing is to change media..please try this protocol dear...
Hope it helps....
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