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.