Dear colleagues: Tell me about your work experience on Limiting dilution method for single cell isolation.
Can I use pen/strep (1%) or I should remove it?
What % of FBS, I add to media?
Hi Zahra I have done quite a bit of limiting dilution on various cell lines (most recently H9C2 and HEK293 transfectants).
In brief use exactly the same conditions as you would for bulk culturing the cells (i.e. if you do not use pen-strep in your T75/T175 flasks do not use it for the single cells). MCF-7 normally grow ok with 10% FBS so use that (or whatever you use in the bulk culture). Also don't forget to include Insulin :)
Some cell lines struggle when diluted to down to the single cell level (I haven't tried MCF7 but I don't anticipate a problem). If your single colonies are not growing you can try using conditioned media (i.e. take some "old" 2-3 day media from a near-confluent flask of MCF-7, dilute 1:1 with fresh media and use this).
Method (all under sterile conditions): Trypsinise and count cells. Dilute to 3-4 cells per ml media. Transfer 100 ul aliquots to wells of a 96 well plate (should get ~1 cell every 3 wells so you may want to plate out 2-3 plates per line). Allow to adhere > 1 hour, Examine under microscope. If you have problems seeing the cells (some cells are pretty hard to see and they can "hide" near the well edge) try Calcein AM labelling and checking under fluorescence (488 nm excitation).
If you can't see any growing colonies after 2 weeks it is likely that it has not worked (in my hands MCF7 have a d.t. of ~ 45 hours although this may vary due to FBS source).
When I have done this in the past I have drawn up a schematic of a 96 well plate (in PowerPoint) printed it out for each plate and marked on this in which well I located a clone, it's rough position and whether or not more than one cell is present in the well (sometimes you get one clone in the top of a well and sometimes another elsewhere).
I think the main problem you will have is actually trypsinising the cells sufficiently to get individual cells. MCF7 grow in clumps generally and are pretty hard to separate from each other without over-triturating the cells and damaging them.
I am not familiar with growing MDA-MB-468 cells but if you ever want to grow SKBR3 don't! SKBR3 has a sister line (AU565) which is derived from the same cancer and grows faster and with less problems so use that instead :)
Anyway, good luck with the clone isolation!
Gary
I have many insect cell line like Sf9, human cell line Hepg2, cancer cell line Hela line.
limiting dilution method requires after screening more and i think FBS concentration will be same you are using for subcultures. But in special cases it will be dependent on the packed cell volume for eg (when using feeder layer).
but finally i think you have to FACS to isolates single cell type
I'm working with human breast cancer cell line (MCF-7 and MDA-MB-468).
Hi Zahra I have done quite a bit of limiting dilution on various cell lines (most recently H9C2 and HEK293 transfectants).
In brief use exactly the same conditions as you would for bulk culturing the cells (i.e. if you do not use pen-strep in your T75/T175 flasks do not use it for the single cells). MCF-7 normally grow ok with 10% FBS so use that (or whatever you use in the bulk culture). Also don't forget to include Insulin :)
Some cell lines struggle when diluted to down to the single cell level (I haven't tried MCF7 but I don't anticipate a problem). If your single colonies are not growing you can try using conditioned media (i.e. take some "old" 2-3 day media from a near-confluent flask of MCF-7, dilute 1:1 with fresh media and use this).
Method (all under sterile conditions): Trypsinise and count cells. Dilute to 3-4 cells per ml media. Transfer 100 ul aliquots to wells of a 96 well plate (should get ~1 cell every 3 wells so you may want to plate out 2-3 plates per line). Allow to adhere > 1 hour, Examine under microscope. If you have problems seeing the cells (some cells are pretty hard to see and they can "hide" near the well edge) try Calcein AM labelling and checking under fluorescence (488 nm excitation).
If you can't see any growing colonies after 2 weeks it is likely that it has not worked (in my hands MCF7 have a d.t. of ~ 45 hours although this may vary due to FBS source).
When I have done this in the past I have drawn up a schematic of a 96 well plate (in PowerPoint) printed it out for each plate and marked on this in which well I located a clone, it's rough position and whether or not more than one cell is present in the well (sometimes you get one clone in the top of a well and sometimes another elsewhere).
I think the main problem you will have is actually trypsinising the cells sufficiently to get individual cells. MCF7 grow in clumps generally and are pretty hard to separate from each other without over-triturating the cells and damaging them.
I am not familiar with growing MDA-MB-468 cells but if you ever want to grow SKBR3 don't! SKBR3 has a sister line (AU565) which is derived from the same cancer and grows faster and with less problems so use that instead :)
Anyway, good luck with the clone isolation!
Gary
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