Growth medium added: 2 ml in each 35 mm plate
20 to 23 microlitre of cell suspension seeded in each plate
In our lab we seed HeLa cells in 35mm dishes for live cell imaging and a good distribution is key for these experiments. What we typically do is to pre-dilute the cells in medium in a Falcon tube (at the desired final concentration) and then add the desired volume into the 35mm dish (typically 2ml). This prevents adding the undiluted cells directly into your dish where each drop might otherwise form a "colony". I try to add the pre-diluted cells dropwise into the dish in order to prevent any kind of vortex. Then I shake the dish in a "cross-like pattern" (forward-backward and left-right) two or three times. Also I try to prevent any spiraling or swirling motion when putting the dish back into the incubator.
In our lab we seed HeLa cells in 35mm dishes for live cell imaging and a good distribution is key for these experiments. What we typically do is to pre-dilute the cells in medium in a Falcon tube (at the desired final concentration) and then add the desired volume into the 35mm dish (typically 2ml). This prevents adding the undiluted cells directly into your dish where each drop might otherwise form a "colony". I try to add the pre-diluted cells dropwise into the dish in order to prevent any kind of vortex. Then I shake the dish in a "cross-like pattern" (forward-backward and left-right) two or three times. Also I try to prevent any spiraling or swirling motion when putting the dish back into the incubator.
Shaking in the cross like pattern is important and then let the plate stand for 1h at room temperature before it goes back in the incubator. This worked at least after seeding in 384 well plates. Good luck!
As everybody else suggested you need to dilute the cells. What we normally do especially when you have multiple samples, count the cells and dilute the relevant number of cells at the appropriate volume that would give you the final desired cell concentration. This way you have homogenous cell distribution in your plates and in between plates. You can leave the plates stand for 20-30min in the hood to allow the cells to adhere and then move to the incubator. Alternatively you can find yourself cell seeders. Here at the University of Glasgow our engineer collaborators Dr P Reynolds and Prof N. Gadegaard have developed either square or round plastic seeders that allow you to seed small volumes of cells in top of anything. They are flat with 2mm high legs for cell seeding space and a whole to allow cell seeding through this.if interested, you can find them at the Glasgow University Scotland web site.
Use flow based cell counters if available or go with the hemocytometer and leave a dilute your cell stocks such that you do not pipette up more than 15-20uL of the cell stock to drop in the PD-35.. Or you can pick up total number of cells required for your experimental setup and reconstitute them into another 50ml conical tube according to containing total media required according to the number of PD-35's you need to set up. You can add a margin of upto 1ml more than required to avoid pipetting error
Hi Anirban,
I have seeded both 3T3-L1 adipocytes and HEK cells in 6-well plates (which is very similar to the 35mm dish). I first count and dilute cells in growth media in a falcon tube, then mix it well gently and add 2 ml to the plate. After that, I also swirl the plate gently in a pattern that looks like the number "8" to make sure cells are distributed evenly on the plate. This works pretty well with my cells. Hope this helps.
Most things having been said about diluting the cells before plating (which is also my method), I warn about letting the cells stand outside of the incubator for longer than absolutely required when working with bicarbonate buffered media: the pH will end up to be about 8 and will take considerable time (> 30 min depending on the volume) to equilibrate back to pH 7.4.
hi Anirban...Johannes has given best option........it is always good to dilute the cells in falcon for equal distribution in 35 mm dish..
Good Luck!
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