Transfection of mRNAs and FACS
Day 1 (cell seeding)
HEK293 cells (wild-type and PEX3 mutant clones) were seeded on a 6-well culture plate (TrueLine) 5 × 105 cell in 2mL / well and cultured at 37℃ 5% CO2 for overnight.
Day 2 (transfection)
The cells were transfected with PTS2-mCherry-PESTmRNA (the translated protein labels peroxisomes when they are present while immediately degrades when there is no or a few peroxisomes by PEST sequence) with EGFP mRNA (labels whole cell) using PEI MAX reagent (Polyscience) and Opti-MEM (ThermoFisherScientific). The cells were cultured for overnight.
Day 4 (FACS)
The cells were washed with PBS, dissociated by trypsin into single cells, and collected in a 15 mL tube with culture medium. After centrifugation (1000rpm 5min, ) cell pellets were resuspended in culture medium and proceed to FACS.
The cells were analyzed by a flow cytometer (Sony SH800). There are three populations in the cell such as an EGFP-positive and mCherry-high population, an EGFP-positive and mCherry-low population, and an EGFP-negative and mCherry-negative (untransfected) population.
The EGFP-positive mCherry-high population and the EGFP-positive mCherry-low population were separately collected by FACS. The collected cells were instantly frozen by liquid nitrogen and stored at negative 80℃ until LC-MS assay.
Transfection and single cell isolation
Day 1 (cell seeding)
PEX3 mutant clone #10 was seeded on a 6-well culture plate (TrueLine) 5 × 105 cell in 2mL / well and cultured at 37℃ 5% CO2 for overnight.
Day 2 (transfection)
The cells were transfected with mCherry-SKLmRNA (labels peroxisome when they are present in the cell while cytoplasm when absent or scarce) with EGFP mRNA (labels whole cell) using PEI MAX reagent (Polyscience) and Opti-MEM (ThermoFisherScientific). The cells were cultured at 37℃ 5% CO2 for overnight.
Day 3 (FACS)
The cells were washed with PBS, dissociated by trypsin into single cells, and collected in a 15 mL tube with culture medium. After centrifugation (1000rpm 5min, ) cell pellets were resuspended in culture medium and proceed to FACS.
The cells were analyzed by a flow cytometer (Sony SH800). Single cells of mCherry and EGFP positive cells were isolated in U-shape bottom 96-well plates (1 cell / well). Subsequently, soritality and the peroxisomal phenotype of each cell was confirmed by a fluorescent microscope (Axioscope, Carl-Zeiss) and photographed (Axiocam, Carl-Zeiss). The cells were independently transferred to a flat-bottom 96-well culture plate and incubated for days to expand the clonal population. After the expansion, each cell clone was immunostained by anti-Catalase and anti-PMP70 antibodies to assess peroxisomal mosaicism.
