Tina,

Our lab found an even plating to be accomplished by moving plates backward and forward, then right to left to right, repeat same motions x 5. Next, allow plates to sit for 3-5 minutes before placing into incubator. We found that any type of swirling would cause the cells to either congregate in the center or along the outside of the wells.

In agreement with Avijeet, typically one would not seed the outer most wells in 24 well and 96 well plates.

When you rotate plates horizontally, cells tend to gather at the center of well. It is suggested shaking a plate back and forth and right-and-left. If you use less medium, more medium stay close to the wall of a well. This could help to avoid that cells to go to the center of a well.

I usually try less medium to seed cells, I rotate plates horizontally first, and then incubate it for 1 min in an incubator. As a result, cells gather at the center of well, but I shake it again in a back-and-forth-and-right-and-left manner this time. The reason I incubate it for 1 min is that cells get down to the surface of the bottom. This means that cells are ready to attach the surface soon. When you distribute cells evenly under this condition, cells attach before circulation of media bring cells to specific locations.

Do the 24-well plates you are using have inserts already? When the inserts come with the plate, usually the top and bottom wells are without inserts. These wells are not to be used, but you dispense HBSS in these wells to keep the moisture in the plate. Before seeding i usually equilibrate the inserts with HBSS for approx. 15 minutes. How much (uL) do you dispense into the wells? What is the cell density? how many cells do you intend to seed per well ? You might want to optimise the cell number per well for your compound of interest. I dispense 200 uL of cell suspension and after I used the cells for experiments, they were visualised using confocal microscope, which then confirmed for the presence of cell monolayers.

It is a common issue due to vibration in incubators. Easiest method to overcome this issue - after adding the cell suspension to wells (minimal volume till cells get attached) gently swirl the plate to spread the cells evenly. Keep the plate on a flat surface for 5 minutes (bench top in the tissue culture room) . Then transfer to an incubator. Also make sure the trays in the incubator are horizontal. Keeping more flasks on one side may tilt the tray towards the heavier side.

Agree with kazuo fushimi u have to shake ur well plate because the diameter for 24 wellplate is quite big.

Easy!...When you plate your cells make movements in a figure of eight, I mean, as if you were picturing an eight...ten times. With this movement I resolved my problem...

I prefer planting at about 50% confluency and allow cells cover the place of the well by dividing and growing

Tina,

Our lab found an even plating to be accomplished by moving plates backward and forward, then right to left to right, repeat same motions x 5. Next, allow plates to sit for 3-5 minutes before placing into incubator. We found that any type of swirling would cause the cells to either congregate in the center or along the outside of the wells.

Mix cell suspension, add to the wells of 24 well plate and leave the plate at horizontal surface at room temperature for 1 hour prior to placing the plate to CO2 incubator. Following this technique should greatly improve even cells distribution across the wells.

As my experiment shaking or "rocking" technique as Kazuo Fushimi and Henry Young mention will be help..

Tina, I believe this question is about the edge effect of the 24-well microplate, right? I am having a same problem and couldn't find any solution to overcome this problem yet. Hopefully someone can come out with some good tips or advise.

Thank you everyone. Leaving the plate on the worktop for a few minutes and doing a figure of 8 motion definatey helped to spread them more evenly.

There several causes for this. Laminar flow hood benches produce vibrations that cause cells to settle unevenly. You don't want the cells cells settling in there if you can avoid it. Incubators also have vibrations. So putting cells too soon into the incubator can be the issue. You may even be encountering convection currents in the media as the temperature equilibrates to the incubator which will move unattached cells around. This may explain the different cell patterns on the edge and center wells of the 24-well plate. These two problems can be solved by settling and attaching cells on the bench for 20 min.

Another possibility is you have poorly coated tissue culture plates. In that case change your supplier. Corning is reliable for having good coatings on tissue culture plates.

Also keep your volume around 0.5ml in 24-well plates. Minimal volumes will give you a ring of denser cells around the edge due the meniscus effect.

Well.....in a simpler manner.....you can 1st add slight medium in the well without cells.....it should cover the surface of well......and after covering the surface of well with medium ....now add your cell suspension into the medium and mix with pipette gently thrice.....it will not attach mid of the well and distribute all over the surface.

I do exactly what Lisandro said. Quick, small , 8-shaped shaking movements will work and spread your cells evenly. It is an easy trick. Try it on the microscope and you'll see :)

As mentioned above by other colleagues, just shake the plate every 10-15 min for the first 60-90 min.

That'll improve your outcome

Doing only figures of 8 did improve the outcome but cells in some of the wells (usually the first few that were seeded) would still group in the center. What worked nicely for me; Holding both the 24 well plate and the tube of suspension in one hand, leaving the other hand free to pipette. Slowly tilting the plate back and forth continuously whilst the cells were seeded to keep them in suspension until I'd finnished. After the 24 wells are filled I leave them on the workbench outside of the hood (to avoid the vibrations) and do several gentle firgures of 8 and leave for 5-10 minutes, then check under the microscope before putting them into the incubator. The cells seemed eager to attach and not setting the plate down under the hood when seeding seemed to make a difference.

Also, putting a piece of tissue down underneath the plate when doing the figure of 8 motions not only avoided that aweful scraping noise but made the movements a lot smoother and so helped to avoid that edge effect that the figure of 8 can give you sometimes.

Mix cell suspension, add to the wells of 24 well plate, move plates backward and forward, then right to left to right, repeat same motions x 5. Next, allow plates to sit for 3-5 minutes before placing into incubator.

I would suggest to make suspension of the cells in complete media and then keep in laminar hood for 10 min and the keep it in incubator this will give u even spreading of cells

Very useful ideas......

One final idea here that builds on the 'figure 8'; after letting cells rest in the hood do your figure 8 distribution directly on the incubator shelf, just leave them when your done. One of the reasons you'll get any residual clustering in the center is due to the motion generated when transporting the cells.

I do basically what Henry Young said. I never swirl to mix multi well plates. I always slide the plate back and forth, left and right about 5 - 10 times in each direction, taking care not to introduce any circular motion into the media. Swirling causes the media to move in a circular motion, and much like sand in a cup of swirling water, it moves the particles to the center. The idea is to limit any type of circular motion. t25 flasks etc with edges don't have this problem, because circular motion of the media is harder to propagate and maintain. Sometimes multi-well plates with square wells work well for this as well. Nunc makes some multiwell TC plates with rectangular wells.

In my Experience in 96 and 6 well plates the most important thing is to get a very good even cell suspension. then seed them without delaying (otherwise the cell will start to sediment or accumulate). After that, shake as figure 8 and slightly left-right and front-back. It works well. Before you incubate check the cells under microscope.

Also, make sure your lab mates don't slam the door of the incubator when cells are freshly plated - if you get wave like patterns, it is likely a vibration issue. Careful not to generate a swirling if you need to walk any distance to the incubator, that will drive the cells to the center, as you would imagine.

Hello everyone,

I have one similar concern regarding tissue culture. Do you think this oscillation movement affects cell migration from the center to the edge of the tissue? I would really appreciate if you forward your view. Thanks

try using EDTA (5uM) to prevent clumping.

When using 24 well plate, I don't use the top and bottom 6 wells, only HBSS or DPBS are added to these wells. Only the middle rows are used for experiments. How many uL and do you load into each well and what is the cell density?

I find pre-incubation of the plates with little amount of media at 37degree very helpful. 

@ Rosalin, I tried seeding by incubating media first and then adding cells to the plate. However, better distribution was achieved by diluting cells outside and adding them directly to a dry plate. It could be one of those cell line to cell line variation. I am working with lung and prostate cancer cell line.