Fitc rat anti mouse igm antibody

Male and female PGCs were isolated from the blood of E2.5 embryos and from the gonads of E4.5 and E6.5 embryos using FACS. Fertilized eggs were incubated at 37 °C under 50–60% relative humidity. Collected gonad cells were dissociated using TrypLE™ Express Enzyme (Thermo Fisher Scientific, Carlsbad, CA, USA). The gonad cells and blood cells were incubated with anti-stage-specific embryonic antigen-1 (SSEA-1) antibody (sc-2170; Santa Cruz Biotechnology, Dallas, TX, USA) diluted with wash buffer (0.5% BSA and 0.1% NaN₃-PBS) at 1:100 for 1 h on ice. After washing with the washing buffer, the secondary antibody reaction was conducted using FITC Rat anti-mouse IgM antibody (553,408; BD Biosciences, Franklin Lakes, NJ, USA) at 1:200 dilution for 30 min on ice. After washing, the samples were incubated with propidium iodide to detect the dead cells. Then the SSEA-1 positive cells were sorted using Cell Sorter MA900 (Sony Biotechnology, San Jose, CA, USA). The sex of each embryo was confirmed using the patterns of chromatin-helicase DNA-binding protein-1 (CHD1) fragment bands obtained through PCR^{56 (link)}. Genomic DNA was purified from a small section of embryos using SimplePrep™ reagent for DNA (TaKaRa Bio, Kusatsu, Japan).

Flow cytometry measurements were performed using a BD Accuri C6 Plus flow cytometer. For the cell cycle assays, cells fixed in 70% ethanol and stored at 4°C were washed in PBS and incubated in PI stain solution (PBS with 1% TritonX, 20 μg/ml RNAse A and 60 μg/ml propidium iodide) for 30 min at 37°C, placed on ice and measured.
For acetylation assays, cells fixed 1% paraformaldehyde (PFA) were stained with Histone H4ac (pan-acetyl) antibody (Active Motif, 39244) and a secondary antibody conjugated to the Alexa 647 fluorophore (Abcam, ab150067).
For class switching assays, CH12F3 fixed in 1% PFA cells were stained with FITC Anti-mouse IgA antibody (BD Biosciences, 559354) and APC Anti-mouse IgM antibody (Affymetrix eBioscience, 17-5790-82), both 1:200 dilution. Primary B cells fixed in 1% PFA were stained with FITC Rat Anti-Mouse IgM antibody (BD PharmingenTM, 553408), 1:200 dilution. Both cell types were incubated with the antibody solutions for 45 min on ice, then washed in PBS and measured.