Preclinical MRGPRX2, Mrgprb2, Mast Cell Activation, and Evans Blue Assays
Calcium mobilization in heterologous cells - Clonal HEK293 cells expressing human MRGPRX2 and Galpha15, created in the lab, were plated in wells of a 96-well plate, loaded with Fluo-4 acetoxymethyl ester (Fluo-4 AM) for 45 minutes at 37°C and allowed to rest for 30 minutes before use, as previously described (McNeil et al., 2015). 2X elamipretide was added by manual pipetting at designated time points, and fluorescence intensity before, during, and after elamipretide incubation was measured with a confocal microscope using the FITC filter. A similar protocol was used for MRGPRX2-transfected Chem-1 cells and their parental cell line, Chem-1. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) high glucose medium (4.5 g/L) with 10% fetal bovine serum, non-essential amino acids, and HEPES. Geneticin (G418) was used to maintain receptor expression in the MRGPRX2 cell line. These cells were seeded to 96-well plates, loaded with Fluo-4 AM for 30 minutes at 37°C, washed, and allowed to rest for 30 minutes before use. Cells were resuspended in 50 µL of PBS with calcium and magnesium and 50 µL of 2X elamipretide or ionomycin were added to stimulate the cells. A no-drug vehicle control and ionomycin positive control were also included. Plates were read every six seconds for two minutes using a Biotek plate reader.
EC50 calculation - Clonal HEK293 cells expressing human MRGPRX2 and Galpha15 were plated in a 96-well plate, loaded with Fluo-4 AM for 45 minutes at 37°C, and allowed to rest for 30 minutes before use. The assay was performed using a fluorescent plate reader and baseline fluorescence was calculated as the average of a 30 second read. 2X elamipretide was added manually after baseline recordings, and response was defined as the maximum signal within 90 seconds after addition of elamipretide minus the baseline fluorescence signal. Concentrations were tested in duplicate, the assay was performed six times, and the curve was calculated as a four-parameter non-linear fit with variable slope.
Peritoneal mast cell activation assay - Primary peritoneal mast cells were isolated from wild type and Mrgprb2 knockout mice (McNeil et al., 2015). These mice were selected because Mrgprb2 is the mouse ortholog of MRGPRX2. Mast cells were incubated in DMEM and supplemented with 10% fetal bovine serum and 100 ng/mL human stem cell factor for 2 hours in a 96 well plate. The cells were then loaded with Fluo-4 AM for 30 minutes at room temperature, washed, and allowed to rest for 30 minutes before use. Using a fluorescence microscope, free intracellular calcium levels in each mast cell were measured. If the Fluo-4 signal rose by at least 50% for ≥10 seconds, cells were identified as responding.
Evans Blue assay - Anesthetized wild type and Mrgprb2 knockout mice up to 8 months of age were injected intravenously with 50 µL of 12.5 mg/mL Evans Blue in saline, followed by injection of 5 µL of 0.5 mg/mL elamipretide in one paw and saline in the other paw. Mice were sacrificed 15 minutes after elamipretide injection and paw tissue was collected, dried for 24 hours at 50°C, and weighed. Evans Blue was extracted by a 24-hour incubation in formamide at 50°C and the optical density was read at 600 nm and 740 nm using a spectrophotometer. The value at 740 nm was subtracted from the value at 600 nm to attain the final readout.