Determination of ADC Cytotoxicity

Cytotoxicity assays are an essential tool to screen promising ADC candidates, predict the in vivo efficacy of ADCs, and evaluate the cell specificity of ADCs.

This article describes how to evaluate the cytotoxicity of ADCs in a monoculture system using the MTT assay and how to evaluate the in vitro bystander effect of ADCs in a co-culture system using green fluorescent protein-transfected Ag-cells, aiming to provide researchers with a comprehensive guide on how to assess the effectiveness of ADCs in killing target cells and bystander cells.

Creative Biolabs typically specializes in providing comprehensive services for ADC development, including in vitro cytotoxicity assays. For detailed information on the specific services offered by Creative Biolabs related to ADC in vitro cytotoxicity assays, please contact us.

Disclaimer

This procedure is only a guideline. Please note that Creative Biolabs is unable to guarantee experimental results if it is operated by the customer.

• Determining Optimal Cell Counts and Incubation Time

Material:
Cell lines (MCF7 and N87 cells)
1640 medium
Dulbecco's phosphate-buffered saline (DPBS), pH 7.4
0.25% trypsin-EDTA
Cell counting machine
Microplate reader
5 mg/mL MTT solution (see Note 1)
10% (w/v) SDS solution with 0.01 M HCL (see Note 2)

Procedure:
1. Prepare a serial dilution of cells from 1 × 10⁶ per mL to 1 × 10³ per mL.
2. Add 100 μL of the dilutions into wells in triplicate, and leave three wells of the medium as a blank control.
3. Incubate the plate at 37 °C for 6-24 h.
4. Add 20 μL of 5 mg/mL MTT solution into each well and incubate at 37 °C for 1-4 h.
5. Add 100 μL of 10% SDS-HCL solution and incubate the plate in the dark at 37 °C overnight (see Note 3).
6. Read the absorbance at 570 nm using a microplate reader.
7. Get the average absorbance values from each cell number group and subtract the average value of the blank. Plot the absorbance vs. cell numbers and determine the initial seeding number for the cytotoxicity assay based on doubling time and the desired duration of the assay.

Note:
1. It must keep each dilution one time more concentrated than the desired concentration in the well.
2. Dissolving SDS will generate bubbles. To prevent these bubbles, avoid heavy vortexing or stirring. Heating can also help dissolve SDS.
3. Using a 10% SDS-HCL solution as a solubilization solution takes more time than DMSO to totally dissolve formazan crystal.

• Monoculture Cytotoxicity Study Using MTT Assay

Material:
Cell lines (MCF7 and N87 cells)
1640 Medium
Dulbecco's phosphate-buffered saline (DPBS), pH 7.4
0.25% Trypsin-EDTA
Microplate reader
5 mg/mL MTT solution

Procedure:
1. Add cells in a 96-well plate at a density of 1000-10,000 cells/well in 50 μL of media volume. Arrange the plate with six main groups: blank medium for Ag+ and Ag- cells, Ag+ control, Ag- control, and ADC-treated antigen positive and negative groups. Treat the "blank" wells with 50 μL fresh medium instead.
2. Incubate the plate at 37 °C with 5% CO₂ overnight.
3. Add 50 μL of prepared ADC solution and fresh medium into each drug treatment well and blank and control wells, respectively.
4. Incubate the plate at 37 °C for 48-144 h (see Note 4).
5. Add 20 μL of 5 mg/mL MTT solution into each well, and incubate at 37 °C for 1-4 h.
6. Add 100 μL of 10% SDS-HCL solution and incubate at 37 °C overnight.
7. Read the absorbance at 570 nm.
8. Calculate the cell viability at different ADC concentrations and plot the data as % viability vs. ADC concentration to get the IC50 value. (see Note 5)

Note:
1. The incubation time depends on the type of toxic payload the ADC has and the cytotoxic mechanism of action for the payload. If the payload is a tubulin inhibitor, 72 or 96 h is better to evaluate the cytotoxicity of these agents.
2. To process the data, the absorbance obtained from all the seeded wells should be subtracted by the average value of blank group. The percentage of live cells is then calculated by dividing the normalized absorbance of ADC-treated groups by the normalized absorbance of the control group.

• Co-Culture Study to Determine the Bystander Effect of ADC

Material:
1640 medium
Dulbecco's phosphate-buffered saline (DPBS), pH 7.4
0.25% trypsin-EDTA
Microplate reader

Procedure:
1. Prepare both Ag+ and green fluorescence protein (GFP)-transfected Ag+ cells.
2. Arrange the plate with seven main groups: blank (background control), Ag-cell only group, and 5 co-culture groups with different antigen-negative cell percentages: 90%, 75%, 50%, 25%, and 10%. Divide the Ag-only and co-culture groups further into two arms: ADC-non-treated (control) and ADC-treated.
3. Add 10,000 cells/well, and ensure a final medium volume of 100 μL, except for the blank group (see Note 6). Add only 100 μL of fresh medium into the blank group.
4. Incubate the plate at 37 °C with 5% CO₂ overnight.
5. Remove the old medium from each well and add 100 μL of ADC-containing medium to each ADC treatment well and 100 μL of fresh medium to control wells (see Note 7).
6. Incubate the plate at 37 °C with 5% CO₂ for 48 h.
7. Read the plate at 485/535 nm (excitation/emission) (see Note 8).
8. Repeat steps 6 and 7 at 96 h and 144 h after ADC treatment.
9. Normalize the fluorescence intensity value in each well by subtracting the reading from the blank group. Divide the fluorescence values of ADC-treated wells with the values of non-treated wells to get % viability (see Note 9).

Note:
1. It requires time to observe the bystander effect compared to the direct cytotoxicity assay. However, it is not advised that adding media during the assay, which will influence the bystander effect by diluting the free payload concentration.
2. It must keep each dilution one time more concentrated than the desired concentration in the well.
3. GFP has a maximum excitation wavelength of 488 nm and a maximum emission wavelength of 510 nm.
4. Compare the viability of the co-culture system with Ag+ cells only. If the difference is statistically significant and less viability is observed in the co-culture system, the ADC molecule has bystander activity.

For Research Use Only. NOT FOR CLINICAL USE.