I want to know about the factors related to (1) while seeding
(2) at incubation
(3) before dosing
Make sure you have done the right cell calculations that you need to seed and while counting the cell number using Haemocytometer, 10 fold dilution of cells using trypan blue is better to calculate to seed the required number to cells in each well. The incubation is at 37 degrees for 24 hours before you have to dose the cells. After 24 hours of incubation make sure to make correct ratio of doses you need to give to the cells which should minimize cell cytotoxicity at the same time, otherwise you may end up with disappointing results.
(1) Seeding - Seed at low density taking into account length of experiment and rate of growth. Your first exp is titrating cell number seeding densities (do not just pick a number based on the literature). Pick lowest cell number you can go - You usually want cells actively dividing during the course of the exposure to drug X and not reaching confluence before the end of your experiment. Make sure you have a single cell suspension - use needle aspiration after trypsin step if required. An example I have used often is 500 cells per well for 72 hrs in 384w format (ie about 48 hrs drug exposure, single dose application). What format is your assay?
(2) At incubation - incubate overnight, at least 16 hours or so. Cells need to recover from the trypsin/seeding step and start to divide again.
(3) Before Dosing - are your cells (a) alive (if adherent cells) and (b) evenly spaced across the well. That's about all I check before dosing.
Do a dose response titration ie 10uM to 1nM over 8-10 data points including DMSO control (or vehicle). Make sure your DMSO concentration is consistent across all doses (eg 0.1%v/v is usually non-toxic to all cells)
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