I have developed a cell-based assay in 96-well plates. After stimulation of mouse neuroblastoma N2a cells with a growth factor I fix them with methanol and measure phosphorylation of Erk1/2 (staining with phospho-antibody against Erk1/2 + fluorescent secondary Ab). The intensity of fluorescent signal is measured in a plate reader. Because I need to use these cells for an automated high throughput screening, I had to switch to the 384-well plate format. Unfortunately, cells detach from the bottom of the plates during washing steps (automatic washer is already set for low speed dispense and aspiration). It has never happened in 96-well plates - the cells stayed attached during all the procedures. My guess is that in smaller wells (384-well plates), the force of meniscus works against cell attachment so they are not as firmly attached as in bigger wells (96-well plates). I only tried Greiner's tissue culture (TC)- and Advanced TC-treated plates. Perhaps I should try other vendors/plastic surface treatments that would warrant the firmest attachment of my cells. Anyone else has the same problem? I would appreciate your suggestions. Thanks!
I think coating the plates with Poly D Lysine plus 2µg/mL L-ornithine should work. Instead of handling with automatic washer if you could do manually, you can keep the plate in slanting position and change the liquids by adding to the wall of the plate, not directly to the cells.
or may be try the ibidi plates, they work quite good
http://ibidi.com/fileadmin/products/labware/plates/P_8840X_Plate_384well/IN_884XX_384.pdf
I was quite unhappy with Greiner plates. You could try BD or sarsted
Unfortunately, I do have to use automatic washer because this assay is designed to screen thousands of compounds so a large number of plates will be processed at a time. Of course I am planning to test all those different coats. I just wanted to save time and hear from someone who already successfuly used 384-well plates for cell-based assays and did not have a problem with cell detachment. Many vendors advertise their plates as perfect for cell-based assays, but reality is quite different from advertisement. Thanks anyway!
We had problems with cell detachment of HEK cells in 24-well plates. Our problem was solved by coating the plates with the already suggested poly-D-lysine plus changing the washing buffer from PBS to HBBS. Changing the buffer had a greater impact in our setting, in case plate coating is not feasable with 384 wells. It seemed that the concentration of certain salts was important (e.g. too much phosphate and too little magnesium in PBS). Good luck!
I have also had success using iBidi plates. They will also send you a free test sample. Since you are culturing neuronal (sort of) cells, and if cost is not too much of an issue, you can try coating the plates with laminin.
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