Colonies are >30-50 cells big after two weeks of culture in methylcellulose based media (Methocult), which is semisolid. I tried to capture individual colonies with sharp pipette tips but it is, apparently, not a very efficient way.
Can you please mention some known methods (syringe etc) or ideas?
Any experience you can share?
Hi Rakesh,
When your colonies are big enough that you can see it by your eyes when you look form bottom of the plate toward light . you can use your sharp marker to draw a point under the colonies. You should try to mark as much colonies as you can. Then wash your plate with PBS and dilute trypsine in PBS 1:10 leave it at RT for 4-5 min and do not move the plate too much please , then try to scrape on the point you have already made and transfer the colonies in a 96 or 48 well plate which you have already added about 200 ul medium. Use p100 and take about 30-50 ul. The cells will be attached over night and you can change the media the following day. In this way in a week or so you will have at lease 10 colonies. when they get around 70% confluency transfer them to bigger 48 or 24 well plate and continue to have each one in 6cm or 10 cm plates. prepare a table in word give each colony a number and write the date you transfer each colony and how fast they grow which will help you for your future experiments with each colony. Be patient and find a way to check each colony to make sure you choose the right ones with the highest expression.
I hope this will help you.
Good Luck,
Nahid
HI Rakesh,
I would let them get a little bigger first. We use a wide bore pipette tip (Fisher catalog# 02-707-134) to punch them out of the gel. The colony can be washed free of methylcellulose and briefly exposed to trypsin (we use accutase, it is more gentle). The single cells should go into sphere culture to maintain them as CSC, if you put them into normal adherent culture they will differentiate quickly as they expand.
Good luck,
Steve
I am using primary cells. So the biggest colonies are not big enough to be seen without microscope.
Do you punch them out while looking under the microscope or you have a way to mark colony area before punching? thanks in advance.
That presents a bigger problem. It is impossible to mark the bottom of the plate while you are looking at it on the scope. We have a small digital microscope in our hood that allows you to pick very small colonies (http://www.bulldog-bio.com/lumascope_iVue.html ) it was $5000. You can easily see and punch out the ones you want and it is sterile in the hood. I think that's your best option. There is also a larger digital microscope (Life Technologies EVOS) that can fit in the hood but those are much more expensive.
Kind regards,
Steve
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