I have to infect them in 24 well plate for an experiment but I see they are already overcrowded (almost confluent), have to do LDH assay after 24 hrs., would it affect my results? Should I go on with my experiment or should I seed fresh cells again?
it should not affect the LDH assay, as in the assay you will have a spontaneous amount of LDH (C-Ve) which should be deducted from all samples depending on the equation that you should follow to calculate the real amount of released LDH.
Overcrowd will be an issue if you use this cell as a culture for infection but for measurement of LDH released in to the media i think it will not be an issue unless your experiment is studying the effect of (some extract or drug--etc) on macrophage in this case you need to be very specify because you doing quantitative measurement for LDH in different dose of your drug or extract and you need same number of healthy-unpacked Macrophage in each well.
Maturation and differentiation status of the cells of myeloid origin are usually affected by the cell density; eventually, it will have an impact on the cell metabolism and certain functions. Therefore, in order to increase the reproducibility of your results, i would recommend to use a fixed concentration of cells. Overcrowded cells will not find enough nutrients and space to proliferate for longer incubation periods and they will also be prone to cell death.
Good luck.
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