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I am currently doing an experiment on cells to test the internalization of a protein. Normally, I seeded my cells the day before the incubation. This worked well for Hela, CHL or PANC1 cells. However, when I did the same with INS1-E and MIN6 (both beta-cells) after the incubation and the washing step the majority of the cells were gone. This was better in the control where I did not put in the compound, but still a lot of cells were detached.

Therefore, I wonder if I should seed the INS1-E and MIN6 cells earlier, more like 2-3 days before. Do these cell lines need more time after splitting to attach again?

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I think that to adequately answer this question, we need to know the composition of your culture medium and any additive with which you supplement it. I'll assume 37 oC and normal oxygen levels. The size of the cultures makes a difference - spinner flasks are not the same as tissue culture plates. –  Larry_Parnell Mar 1 '12 at 13:36
    
The common formulation for INS-1 cells is to use RPMI media, and it's an adherent cell line. I don't know about MIN6. –  leonardo Mar 12 '12 at 12:04
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I've worked with MIN6 cells in the past, and have found that they can take at least 12-24 hours to fully seed (if they are being cultured on polystyrine). Depending on the specifics of your experiment, and whether doing so would complicate your controls, you might also want to try coating the surface you are attempting to seed on with laminin. MIN6 cells seem to like growing on that over uncoated polystyrine and other ECM proteins.

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Searching literature to see what the published protocols were is the best bet. I found a simple document for INS-1 cells here. There is also this paper describing the isolation of an Ins-1-derived cell line 832/13 which my lab (though not me personally) have experience with. Cells should only be sub-cultured when nearly or fully confluent. With Ins-1 cells that would be a sub-culture ratio of 1:4 to 1:8 and will take 2-3 days. Something to note is that Ins-1 cells are poorly adherent to tissue culture plastic. That's a normal part of their characterization. I remember lab members having to take special precautions with respect to media ingredients to prevent them from detaching, as well as really delicately adding media to the dishes otherwise the force from pipetting too quickly will dislodge many cells and explain why your cells are washing away.

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