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I am doing an experiment where I have treat the cells with a drug and calculate their counts. I would like to know if is bad to trypsinize the cells in consecutive days i.e. twice within 48 hours. How much time do the cells need to attach again on the surface of the flask? I want to know if I can treat them with drug the same day I add trypsin for counting.

Thanks.

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  • $\begingroup$ Your question was unclear and I edited it a bit. See if this is what you really wanted to ask. $\endgroup$ – WYSIWYG Jun 15 '15 at 7:29
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    $\begingroup$ Can you specify the cell strain? It really depends on it. I have done for example trypsinization on RPE1 cells (human fibroblasts) twice in 48h without troubles. $\endgroup$ – alec_djinn Jun 16 '15 at 13:40
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It would be stressful for cells to be trypsinized 24 hours after seeding. After cells are plated, there is a lag time to start growing. Perhaps, during the lag time, physiological state of cells is stabilized. They have to express proteins digested by trypsin and display on the surface etc. Then you could disturb the state again by trypsinizing cells again if you replate cells after 24 hours. I would not deny some cells are not affected by trypsin much, but you should be carful.

if I can treat them with drug the same day I add trypsin for counting.

It is not preferable, but in some cases, such a way is taken actually. If you could, you might want to give us more information about which cells and drugs you are going to use.

PS

If you trypsinize cells to count and do not replate them, you do not need to worry about the cell condition, as WYSIWYG says.

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It is not advisable to trypsinize the cells too often when you have to maintain them. However, in your case the situation is different.

Time required to attach depends on cell type. In any case while performing an experiment, your cells should not be already under stress. Therefore you should seed the flasks/culture plates with a smaller concentration of cells such that they reach around 60% confluence in 24 hours. 24 hours is generally considered to be enough for cells to recover from stress due to trypsinization. (You can have a look at this post for doubling times of some cell lines).

After doing the treatment you can count cells whenever you think it is fine; this actually depends on your experiment and you can treat the cells for the time that suits the drug's pharmacokinetcs. Trypsinizing is fine now because you won't be using these cells again.

However, there are methods to calculate cell numbers without trypsinizing. Assuming that your flask is homogeneous, you can choose a section of it and calculate cell numbers manually using microscope. You may also use software like ImageJ to make the task easier. GFP expressing cells will also make the task easier.

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Might be considered "not answering the question" but:

Option 1: Depending on the type of cells you are using, you can use milder methods to detach cells. Example include Versene which is essentially PBS + EDTA. Milder still is to just use PBS that has no Mg or Ca (no chelation).

Option 2: Duplicate your setup - instead of two dishes - one with drug, one without, you make 4 dishes (dispensed from a common source - i.e. if your dish is a 6cm dish with 3ml media, you make 4.5x3mls of cells at the final dilution you want) each plate gets 3mls from the same source so you know they have the same number of cells. Two dishes you add drugs, two you don't. On the day you want to count, you take the replicate plates and count them. This way, you get the count without touching the final dishes at all.

As a side experiment, if you put those trypsinized cells back on two dish (and replace the drug media in the drug treated replicate), and you count all plates on the last day of your experiment, provided cells are not at 100% confluency, any difference you see from your trypsinized vs untrypsinized can be viewed as potential error that you would have introduced if you went through your original methodology.

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