Calcein AM Release Assay to Measure Complement-Dependent Cytotoxicity
In order for the complement cascade to be activated, the circulating complement protein C1q must first be recruited to the cell surface by the bound immunoglobulins on the tumor cell surface. The initiation of the complement cascade leads to the downstream formation of the membrane attack complex. The membrane attack complex forms a pore on the cell surface and thereby permeabilizes the cell membrane which leads to cell death.
(Adapted from) Paul Carter. (2001) Improving the efficacy of antibody-based cancer therapies. Nature Reviews Cancer 1:11, 118-129
By staining the target tumor cells with cytoplasmic dye calcein AM, we can monitor cell death by examining the retention of calcein AM within the cell. Only live tumor cells with intact cell membranes will retain the green dye and while the dead cells will release the calcein into the surrounding media. Based on the retention of the dye the Celigo software can enumerate the number of live target tumor cells over time.
Measure CDC using Direct Cell Counting of Calcein AM Stained Target Cells
Celigo Experimental Protocol
Target cells (adherent or suspension) were collected and stained with calcein AM
Target cells were seeded in the wells of microplates
Complements were added to the wells
Antibodies were added to the wells at different concentrations
The wells were scanned and analyzed using Celigo for direct cell counting calcein AM stained target cells
By observing the reduction in target cell number over time we can determine the percent cytotoxicity for the complement dependent cytotoxicity assay.
Dose-Dependent CDC Images
As time and antibody concentration increased, the number of calcein AM stained target cells decreased
Low [antibody], lower cytotoxicity is shown
High [antibody], high cytotoxicity in shown, where almost 100% of the cells lost the calcein AM fluorescence