Taking Culture Medium Samples for LDH Assay - (Oct/17/2012 )

Hello, I have a disagreement with a colleague on what method is the best for assaying LDH release in cells in culture. The cells are adherant and are cultured in 96 well plates before being assayed. The first method is to remove the aliquot for assaying from the top of the media in the well, with the thought that as long as you are taking it from the same location in each well, you can compare well to well. In the instance each well is one N, so if I want 3 N's I would run 3 wells per treatment. The second method is to remove double the volume from the well, place it in an eppendorf tube, pipette up and down to mix it up, and place half of the volume into 2 different wells. The assay would be set up with 2 wells per treatment, and 4 resulting wells to assay for LDH. Which is the more appropriate method for assaying LDH release by the cells, and not testing the fidelity of the LDH kit? (I also have the appropriate positive and negative controls for total LDH release included in the assay).

Hello, I have a disagreement with a colleague on what method is the best for assaying LDH release in cells in culture. The cells are adherant and are cultured in 96 well plates before being assayed. The first method is to remove the aliquot for assaying from the top of the media in the well, with the thought that as long as you are taking it from the same location in each well, you can compare well to well. In the instance each well is one N, so if I want 3 N's I would run 3 wells per treatment. The second method is to remove double the volume from the well, place it in an eppendorf tube, pipette up and down to mix it up, and place half of the volume into 2 different wells. The assay would be set up with 2 wells per treatment, and 4 resulting wells to assay for LDH. Which is the more appropriate method for assaying LDH release by the cells, and not testing the fidelity of the LDH kit? (I also have the appropriate positive and negative controls for total LDH release included in the assay).


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If what I understood is correct, in the first case you will have 3 separate wells per sample and take say 100 micro liters from each well and use them separately for LDH (finally you will have 3 wells of 100 micro liter each). In the latter case, you have 2 wells, take 200 micro liter from each well and then split each 200 micro liter into 2 and end up having 4 wells of 100 micro liter each.

If that is what you meant, I believe the first method is the better one as it will give you 3 independent values. The second one will give you 4 values but those 4 are not really independent of each other.