Real-time Cytotoxicity Assays in Human Whole Blood

The aim of this procedure is to measure cytotoxicity of target cancer cells in human whole blood with single cell resolution First, a single cell array of target cancer cells is generated. Human whole blood is added to the cell array and drugs such as antibodies are applied. The cells are then incubated with a cell viability dye, such as propidium, iodide, and high throughput imaging is used to measure the number of dead target cancer cells stained red, ultimately providing drug cytotoxicity efficacy.

The main advantage of this technology over existing method, such as LDH and chromium assay, is that cancer cell killing can be done in human whole blood with imaging capability. This method can evaluate antibody cytotoxicity in vitro before animal study Begin by obtaining cell attachment 96. Well plate kits or single cell rate eight well chamber slides here, the 96 well plate will be used.

Apply 1.2 microliters of the activator to the tube containing DNA reagent from the kit, cap the vial, invert it and gently shake to mix the solution. Allow the solution to react for 20 minutes of room temperature. In the meantime, prepare the target cells.

In this demonstration, Raji lymphoma cells will serve as the target cells. Maintain them in supplemented RPMI 1640 culture medium, A 37 degrees Celsius in a CO2 incubator. To prepare the target rahi cells for the experiment, transfer five to 10 million of them to a 15 milliliter conical tube centrifuge for three minutes of 468 times G.Following the spin, use a pipette to aspirate any bubbles that formed on the surface of the supinate.

Then remove the entire supinate and discard it. Next, add 10 milliliters of PBS to the tube. Resuspend the cells by pursing the solution up and down to break up the cell clump, centrifuge the cells again for three minutes of 468 times G after the centrifugation, aspirate any bubbles that formed on the surface of the supine agent and remove and discard the entire supine agent as before, after the activator and DNA solutions from the kit have incubated for 20 minutes.

Apply the mixed solution to the target cell pal in the 15 milliliter tube. Pipette the solution to break up the cell pellet. Next, add green fluorescent cytosolic dyes at a final concentration of 500 Nanomolar.

Place the target cells with the mixed solution and the dye on an orbital shaker and incubate for 30 minutes at room temperature. Following the incubation, wash the cells twice in five milliliters of PBS, centrifuging between washers and resuspend. The cells in the appropriate volume of RPMI 1640.

Growth medium to bring the cell concentration to five times 10 to the six cells per milliliter. Next, apply 200 microliters of the cell solution to each well and incubate for 10 to 15 minutes of room temperature. Following the incubation cells are dear to the spots lightly shake the well plate and look for a raise.

Then gently wash away the unbound cells by pipetting up and down. Then remove the original medium and add new medium to perform the whole blood cytotoxicity assay. Remove the medium in each well and then add 180 microliters of human whole blood to each well.

Next, starting with five milligrams per milliliter of anti CD 20 antibody. Perform serial dilution in 1.5 milliliter tubes to make fifty twenty ten five two one, and 0.5 micrograms per milliliter. Then add 20 microliters of each concentration to each sample.

In triplicate. Add propidium iodide as a final concentration of 500 nanomolar to each. Well then incubate the plate at 37 degrees Celsius with 5%carbon dioxide for 16 hours.

The next day image the cells with an automatic imaging system using atex magnification. Use the cell counting program of the automatic imaging system to quantify the results.Here. The use of cell reporter software is demonstrated.

This software provides simple step-by-step operations for cell counting. First, from the main page of the software, select review results. Then select the sample file.

When the file is open, select the fitsy mean intensity gate to greater than 50, and the Texas red mean intensity to greater than 20. Next, in the toolbar select plate overview, the plate layout and the percentage of cells stained by PI will be shown on the screen. Select export tables.

The system will ask where to save this Excel file, indicate the location and save the file. Finally, import the data to Microsoft Excel. Then use the formula shown here to the percentage of target cancer cell killing to demonstrate the whole blood cytotoxicity assay, anti CD 20 antibodies and the lymphoma cell lines.

Raji, which have a high copy number of CD 20 on the cell surface and mc car, which have a low copy number of CD 20 were chosen as a model system. The detection of the targeted Raji lymphoma cells, both alive and dead was performed as described in this video. When 2.5 micrograms per milliliter of anti CD 20 antibody was used, the level of cytotoxicity was high as indicated by the ratio of dead cells shown in red and green to presented cells shown in green only the level of cytotoxicity decreased as the concentration of anti CD 20 antibody decreased in concentration.

When 0.05 micrograms per milliliter of anti CD 20 antibody was used, the level of cytotoxicity was low. All hundred data points per antibody concentration were analyzed for each antibody concentration. The percentage of death of Raji lymphoma cells was significantly higher than that of mc car control cells.

An effective concentration or EC 50 value of 0.12 micrograms per milliliter was obtained for the whole blood cytotoxicity assay result against Raji. This value indicates that at least 0.12 micrograms, a anti CD 20 antibody is needed to kill 50%of CD 20 positive lymphoma cells in the context of human whole blood. This provides the drug efficacy and dosage needed for use in animal studies and human clinical trials.

Following this procedure, drug resistant cancer cells can be identified and isolated for further analysis. After watching this video, you should have good understanding how to measure cytotoxicity of target cancer cells in human whole blood.