A very obvious one - but is there plenty of water in the water tray of your incubator?

I've had cells dying off for "no reason" before - only for someone to point out the dry water tray!

Sometimes it is bad batch of the plates, in which random wells are not suitable for cells to grow. Once happen to me with falcon plates.

First of all thanks for your answers.

Philip, I thought the same thing with you. I've had checked the water level in the incubator and the level was max level as it should be. Water level is checked routinely in our lab.

I used different brand of plates such as Falcon, VWR, Greiner but the result did not change. As I mentioned before I have used this protocol so I know they work. Trouble started when we moved to other building.

Dear Aylin Ozkan,

Do you share the incubator with others or is it for your sole use? How often and for what duration is the door open on your incubator? Is there any difference with respect to placement of your cultures on different shelves of your incubator, how close or farther the plates are from the water source? Have you tried different lot numbers of plates?

Depending on cells you are trying to grow, especially on substrate-coated plates, sometimes the outside wells are not coated for whatever reason. Switching lot numbers of plates may correct your problem. Switching supplier of plates may also correct your problem. For example, when I began choosing the different culture vessels we used for our studies we empirically tested culture ware from many different companies. What we discovered for our cells was that Falcon had the best T-25 and T-75 flasks; Corning had the best 6-well, 24-well, and 48-well plates; and Costar had the best 96-well plates. Seems that these companies used different plastics depending on the culture vessel being produced. It may occur similarly for the ability to retain moisture or not within the 96-well plates. Something worth trying if you keep having the problem.

Take care,

Dr. Young

Hi Aylin,

I guess it must be something about the new building or the new incubator then if you see the same with multiple plate manufacturers. Do you share the incubator? If your new labmates are leaving the incubator door open for long periods of time that could be an issue.

Is there another incubator in another lab you could borrow some space in for a few days? Preferably one that is not heavily used. Then set up two plates - one for each incubator.

Is there any kind of heavy activity in the rooms around (or in your lab) that might be affecting your incubator?

When I was at Warwick, our cells would always grow in rings in multiwell plates, but only during the summer. Eventually we worked out that a piece of equipment for the building air cooling was directly above the cell culture lab, and subtly shaking the incubators.

In our experience, another factor that sometimes can affect cell growth in plates or petri dishes, in particular, is the ‘edge effect’. The ‘edge effect’ affecting the top plate/petri dish containing cells can be minimized by placing an extra plate or petri dish containing sterile PBS/water/medium on the top. For 24-96 well plates, cells can be plated in the middle wells leaving one row of wells on four sides for sterile PBS/water/medium. These steps can help improve the cell growth, especially when working with low number of cells.

Thanks you for your answers.

Dr. Young, I do not share the incubator with others therefore there is no continuous door opening and closing. The same problem was observed in the plate at the bottom shelf although the shelf is closest to the water tray. This problem was observed in the plate I was constantly working on. The lot number had not been changed when the problem started. I do not think that the problem is the plate because the problem occurred on the same plate. Trouble started when we moved to other building. I used the same materials in the new building. Thank you again for the useful information about the choosing the best plate/flask.

Philip, I will check the air conditioning system. The system may have affected because there is ventilation in the area of the incubator at the room. I didn't fully understand how the cells grow in the plate when there was a air cooling problem.

Nasim, the results were good although I did not add an additional water-filled petri dish, so far. If I would have done the experiment for the first time, your suggestion could be done as you said for the evaporation problem.

Dear researcher

I suggest to replicate your culture in three situation

1. do the same procedure with extra control

2. decrease the volume of medium gradually

3. change your plate with new batch number

good luke

Dear Aylin Ozkan,

I have faced the same problem several times, so, I suggest you to:

1. Use new 96-well plate.

2. Try to reduce the cell concentration to 8000 cell/well if they are continous cell line or 10000 cell/well if the are primary cells.

3. use a inactivated FBS at 2% of the final medium in the wells.

I hope this will help you.

Hi Aylin,

The phenomenon is called the "edge effect" in which a certain amount of fluid evaporate from the outer wells. It affect growth of the cells and as a consequence, give absorbance reading that affect your expected results. I overcome this by avoiding the outer wells (I only place PBS in these wells to minimized potential evaporation from the inner wells). However, this may not always seem feasible in high-through put assays.

Theremofisher appears to have a type of plate that may be helpful (if budget allows this):

Check out the link from Thermofisher for more info:

https://www.thermofisher.com/blog/cellculture/reducing-the-edge-effect/

This link takes you to a 96-well plate designed with a moat around the edge, to add water or other liquid to avoid evaporation:

https://www.thermofisher.com/order/catalog/product/167425?cid=fl-cmd-edgeplate&kui=vMoOmCEEkdm-Njo_tes7WQ#/_ts=1467063983627

Good luck.