An anti-cancer drug or antibody drug conjugate (ADC) screening assay is the first step to establish the utility of a drug candidate in killing cancer cells. Nevertheless, these assays are time consuming and tedious. The purpose of this article is to make things easier when you are required to set up these in vitro screening assays.
How to set up your drug screening assay
To simplify, the drug or ADC analysis process can be divided into 3 steps:
Step 1: Plate Your Cells
Carefully select the target cells to test the efficacy of your compound. Usually, cancer cell lines (tissue specific tumor types, drug resistant/sensitive cancers) are used for drug screening.
Use the 96 well plate format as it works the best to cover a broad concentration range.
Keep your plating density to 5000-20,000 cells in 200µl medium/well. The plating density will vary depending on cell growth and time of incubation [24, 48 or 72h].
Use reservoir trays for preparing large volumes of cell dilutions and subsequent plating.
One 96 well plate can cover testing of two drugs in quadruplicate (Drug1: rows A-D, Column 2-12; Drug2: rows E-H, Column 2-12). Keep common negative (rows A-D, column 1) and positive (rows E-H, column 1) controls for both drugs.
Wait overnight after plating your cells before you treat with the compound.
Plate 1: Overview of cell plating strategy for drug screening assays in 96 well plates. Transfer 5000-20,000 cells in 200µl medium to the wells of a 96 well plate (marked in green). Each plate contains four negative control wells [marked in pink, left empty; wells 1(A-D)]. Later, wells 1(E-H) will act as no drug positive control wells [100% cells survival]. Incubate the plate at 37ºC, 5% CO2 overnight before adding the drug dilutions.
Step 2: Prepare Drug Dilutions
This step requires calculations (of course you can calculate!!). You need to decide the testing range of your compound. This is usually based on previous reports related to the compound`s anticancer efficiency/toxicity range. If you are the first one to test the compound, simply test it first at 3 broad concentrations (say 100µM, 10µM and 1µM).
Once you know the crude range, decide the highest concentration you will expose the cancer cells to. Multiply this concentration by two. This is your working concentration (e.g. if 10 µM is the highest concentration for your experiment, 10X2= 20 µM is the working concentration). You will need around 0.8 ml volume of this for quadruplicate testing.
Prepare a 2 fold serial dilution from this working concentration in a 96 well plate. Multi-channel pipettes are a life-saver for this step!
Plate 2: This model shows serial dilution using 2 fold dilution method that can test 2 compounds in a single 96 well plate. In column 1 (marked in red), add 100µl medium . In column 2 (A-D, marked in green as 1), add 200µl of compound 1 working concentration. In column 2 (E-H, marked in green as 2), add 200 µl of compound 2 working concentration. In rest of the wells (marked in yellow), add 100 µl of medium. Use a multi-channel pipette to transfer 100 µl solution from column 2 to column 3 and mix about 3 times. Repeat this step for all subsequent columns until you reach column 12 (will contain 200 µl final volume).
Step 3: Drug Your Cells
Remove the entire medium from the plated cells in Plate 1. Using a multi-channel pipette, transfer 95µl fresh medium to each well of the 96 well plate. Then, transfer 95µl of the above dilutions from Plate 2 into Plate 1 into the same wells. This will give you both negative blank (A1, B1, C1, D1) and positive, no drug controls (E1, F1, G1, H1) on the plate. Incubate the cells for required time points.
Once incubation is complete, use cell viability, cytotoxicity and/or efficacy assays for ADC characterization to test the efficiency of your drug candidate, which I will discuss in my subsequent article.
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