Filter Paper Method

Some cell lines are difficult to clone by seeding at low density. They seem to need growth factors released by other cells in culture to divide. If you are having trouble cloning your cell line by seeding at super low density, try seeding several 15 cm dishes with 200, 400, 800 cells. Once you get a plate with a nice amount of healthy colonies growing on the bottom (that are well separated from one another) take a 3 hole puncher and punch a bunch of holes out of some filter paper. Autoclave the punches in a container and then do the following:

1) mark the colonies in your 15cm dish that look favorable (good GFP signal etc...) using a dot on the underside of the plate

2) wash your 15cm plate 2x in PBS and then suck off all of the PBS

2) Using sterile tweezers, dip a piece of filter paper into Trypsin and then lay the filter paper ON TOP of the colony so that the filter paper holds a small bead of trypsin over the colony of interest.

3) place your plate back in the incubator for a few minutes to allow the colony to detach

4) Remove the filter paper and place it into wells of a 12 or 24 well dish with media ( many of the cells will stick to the filter paper and thus be transferred to the new dish)

After a day, you can change the media and remove the filter paper. your cells should be attached to the bottom.

good luck

- Trent

I think first dilution followed by FACS will help to get single clones. Allow the cells to proliferate so that proportionality fluorescence increases in clones then sort them probability of getting single clone is maximum.

We have also just carried out a similar experiment using CRISPR and have isolated single colonies.

We use ClonaCell-TCS medium from stem cell technologies.

http://www.stemcell.com/en/Products/All-Products/ClonaCellTCS-Medium.aspx

This has worked very well. After transfection we plate out into 6 well dishes for 1000, 100 and 10 cells per well. After 7-10 days there are some nice single colonies that can be picked using a P200 pipette into a 96 well plate and then expanded from there.

If your cells are adherent, then they may still work with this medium as well.

We have tried sorting and yes, the efficiency of getting a single cell per well is very low and most do not survive.

Otherwise, we did serial dilution as well to get 1/2 a cell per well of a 96 well plate. This means that very second well should have one cell.

All the best.

Filter Paper Method

Some cell lines are difficult to clone by seeding at low density. They seem to need growth factors released by other cells in culture to divide. If you are having trouble cloning your cell line by seeding at super low density, try seeding several 15 cm dishes with 200, 400, 800 cells. Once you get a plate with a nice amount of healthy colonies growing on the bottom (that are well separated from one another) take a 3 hole puncher and punch a bunch of holes out of some filter paper. Autoclave the punches in a container and then do the following:

1) mark the colonies in your 15cm dish that look favorable (good GFP signal etc...) using a dot on the underside of the plate

2) wash your 15cm plate 2x in PBS and then suck off all of the PBS

2) Using sterile tweezers, dip a piece of filter paper into Trypsin and then lay the filter paper ON TOP of the colony so that the filter paper holds a small bead of trypsin over the colony of interest.

3) place your plate back in the incubator for a few minutes to allow the colony to detach

4) Remove the filter paper and place it into wells of a 12 or 24 well dish with media ( many of the cells will stick to the filter paper and thus be transferred to the new dish)

After a day, you can change the media and remove the filter paper. your cells should be attached to the bottom.

good luck

- Trent

You can also try using the spent media (alone or in ratios with fresh medium) by the same cells, so that you get the factors secreted by other cells to keep your single cell clone healthy.

Can you see the fluorescence if you grow them on a 15 cm plate in colonies like Trent suggested? You can mark the colonies that are GFP positive. I then like to use cloning cylinders and a bit of grease to pick colonies. Once the colonies are visible you can put an autoclaved cylinder with a little bit of autoclaved grease around the colony and pick them with a bit of trypsin and a p200 into a 96 or 24 well plate (dependent on your cell type). I buy the cylinders and grease from Sigma.

Going off slightly into the bushes....The need for autocrine growth factors is often a limitation in single cell cloning. The suggestions of using spent media ( 25-50%) is a good one . Many years ago we used to use "feeder cells" that were UV irradiated or Mitomycin C treated to provide these factors. I guess you could also use a transwell insert in a 6/12 well plate with autologous cell in the insert to provide a source of cytokines/growth factors if the growth factors are labile. Has anybody tried growing adherent colonies on plastic/ acetate or Mylar sheets cut, aligned and placed in the bottom of a tissue culture dish. Identified colonies could be excised with a scalpel and transferred to another vessel - trypsinised etc -just a thought before coffee....

"I don't get any fluorescent cell that survives long enough to do the clonal expansion"...how long do you wait post-transfection before trying FACS sorting? Is it possible that your main current problem is transfection efficiency?

Otherwise, I have nice expirience with FACS sorting and propagating of clones with not that "easy" cells. One important thing (maybe someone already mentioned it) is the serum in your cell culture medium....there are serums and serums and even one company serum vary quite from charge to charge...

I recently published a paper describing a really convenient way to isolate live single cells for continued culture with fluorescence confirmation. In the paper we lyse the cells to do RT-qPCR after incubation, but you could just as easily continue culture, allow a colony to grow and then move them to a larger culture vessel.

The benefit to the method is really small volume culture, so the cells will quickly condition their medium. I suppose that is why 96 well plate isolation is so difficult. Also, it's really fast to identify wells that contain a single cell.

The method is described as a very step-wise protocol that may be useful to you. The consumable, a 72-well Terasaki plate, is just a few dollars (you can buy a pack from VWR or Corning).

Flow sorting even on robust cells can be highly stressful and the high voltage chamber it runs through can quickly bleach fluorescent proteins. This method allows normal, aseptic culture techniques for obtaining single, live clones.

I've referenced the paper here.

Article A convenient, optimized pipeline for isolation, fluorescence...

Not an answer, but a related question - when I try to visualize GFP+ cells in transiently transfected cultures, I see a ubiquitous green background.  I tried gently washing cells with warm DPBS+Ca+Mg to get rid of phenol red background, but still have a bright green field.  Thanks for all the other suggestions.

Kris, can you explain more about your imaging setup? What sort of microscope? What sort of culture vessels? Transfection method? Cell type?

We have a Zeiss Axiovert.  I typically use the 10X or 20X objective, with a GFP filter.  These are VK2 E6/E7 (epithelial cells) grown in Keratinocyte Serum Free Medium in 100 mm TC plates.  I'm transfecting them with a pair of double nickase plasmids, using Mirus TransIT-X2.  A coworker told me to be sure the lamp was completely warmed up, and that helped. 

Hello Trent Place, do you have a published reference for the filter paper method?, I will like to use the Filter paper method.

I have also had success using filtered spent media (we call it "conditioned media" in our lab; 33-50%) when isolating individual cells for cloning.

Should we perform centrifugation after trypnisization to replate cells after then in 96 wells plate!

After transfection, you can select recombinant clones in different ways. 1. If you have an antibiotic resistance gene in your vector, you can isolate the recombinant clones by adding antibiotic to the culture medium followed by Filter Paper Method or Scratching Method. 2. If you have a GFP gene in your vector, you can isolate the recombinant cells by FACS technique. 3. If you do not have any of these factors in your vector, you can isolate your recombinant vector randomly by creating a proper cell dilution in 96-well plates followed by a PCR.