Hi everyone,
I'm trying to do a limiting dilution and isolate one clone from T24 cancer cells after a gene knockout.
I followed these steps:
First, used a hemocytometer to quantitate the homogenized cell solution.
Homogenized cell solution concentration: 5.45 × 10^6 cells/mL
To seed one 96-well plate, made 10 mL of a 10 cell/mL solution (1 cell/well). Calculate the total cells needed:
Total cells needed: 10 mL × 10 cells/mL = 100 cells
Determined the volume of homogenized cell solution that corresponds to 100 cells:
Volume of homogenized cell solution needed: (100 cells)/(5.45 × 10^6 cells/mL) = 0.01 µL
Because this is such a small volume, first I made 1 mL of a 1:100 dilution of the homogenized cell solution by adding 10 µL of homogenized cell solution to 990 µL complete medium. Instead of transferring 0.01µL, I can transfer 100 times that volume, which is 1µL.
Since I was planning to do 5 plates, I added 5 µL (of a 1:100 dilution) to 50 ml.
After seeding the cells on the plates, I checked the plates under a microscope, but all the wells I checked were empty, and I could not locolize one cell per well. So I'm suspicious about the methodology.
If someone tried this technique before, I would like to know:
1. If the steps done are correct
2. If there is another technique or trick for making this experiment work (Limiting dilution and single cell cloning),
Thank you!!!
In theory you should end up with 1 cell per well when doing limiting dilution. In reality, you sometimes end up with anywhere from 0-4 cells per well. Just so you are aware that does happen and it is far from a perfect technique.
One problematic area is counting cells on a hemocytometer. Make sure you are counting the large areas in each of the corners of the grid and then take the average of the four. You may want to do this say 3 times (3 different aliquots of cells) so you get a good idea of what the actual concentration is. If your count is falsely high then you may end up with no cells at the end.
To check this you can start with a higher concentration of cells per volume (10-20 cells/100ul) and then aliquot into a few wells and then see how many you end up with. If that looks good then you can dilute them down such that you get 1 cell/well. You did not mention what volume you aliquoted the cells into the wells in but you should aliquot large volumes (50-100ul) since larger volumes are more accurate than smaller volumes.
Good luck!
I don't know about T24 cells specifically but in addition to what Tom Masi said, some cells are prone to dying from sheer stress and you have to avoid doing rough handling when diluting them down, including doing gentle mixing (gentle inversion rather than pipetting up and down) and using wider bore pipette tips with bigger volumes - ie. dilute 1:100 by adding 0.5 mL cells to 50 mL media, so they aren't subjected to the mechanical stress of being forced through a narrow pipette tip.
Some cells also have a tendency to die when diluted down to very low densities regardless of how gently they're handled during dilution. You can sometimes improve their survival by taking media from an actively growing sub-confluent flask of the same cell line (in your case, T24), filter sterilizing it, and diluting it 1/2 or 1/3 with fresh media and using this to seed and feed your clones instead of using 100% "non-conditioned" media. The idea is that you can potentially capture some soluble growth factors and metabolites that normally promote the survival of neighboring cells without actually needing neighboring cells themselves.
Another option is to use a feeder cell layer (usually fibroblasts) that have been mitomycin C or radiation treated so that they don't replicate on passage but are still viable for a few weeks and secreting extracellular matrix/growth factors to promote survival for your clones. The caveat with the feeder cell option is if they aren't fully rendered non-replicative by the treatment they can contaminate your "clonal" cell line, and if you need to maintain antibiotic selection pressure on your clones, the feeder cells can die early from the antibiotic and fail to synthesize required growth factors.
Tom Masi
Thank you for your reply.
1. For the hemocytometer part, I did count the average of 2 chips, and the number I mentioned was the average of both, as they were close to each other.
2. For the volume, I aliquoted 100 µl/well in a 96-well plate.
Finally, yes, it is far from a perfect technique, as some wells were empty and others had 2-3 cells. As I'm performing this for the first time, I was a little bit skeptical about it.
@Alexandra Johnson
Thank you for your reply and provided information.
For the T24 cell line, it is an epithelial cell line of urinary bladder carcinoma. From previous cultures, I can judge that the cell is as resistant to stress as most cancer cells. But since I'm using a KO cell this time, I hope things goes well. I’ll consider these details and implement them if required, in the future.
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