Alexa fluor 594 conjugated anti mouse igg antibody

Cultured WJ-MSC were visualized with bright field microscopy (Leica DFC 300 FX, Leica Microsystems, Wetzlar, Germany). For the analysis of cell surface markers, WJ-MSC were seeded in 2-well chamber slides at a density of 2000 cells/cm². Upon adherence, cells were fixed with 4% paraformaldehyde (PFA) in PBS, blocked with PBS containing 1% bovine serum albumin (BSA; Merck KGaA, Darmstadt, Germany) and stained with fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal antibodies against CD90 (11-0909-42, Thermo Fisher Scientific Inc.) and CD45 (550539, BD Biosciences, Franklin Lakes, NJ, USA), and with a polyclonal rabbit antibody against CD73 (550256, BD Biosciences Inc.). The antibodies were diluted 1:100 in blocking buffer and incubated overnight. For the detection of the unconjugated antibody against CD73, the cells were incubated the next day with an Alexa-Fluor 594-conjugated anti-mouse IgG antibody (1:1000, a11005, Thermo Fisher Scientific Inc.) for 1 h at room temperature. Stained WJ-MSC were analysed using a fluorescent microscope (Leica CTR 6000).

Indirect immunofluorescence microscopy was performed as described previously (70 (link)), using rabbit anti-FLAG polyclonal antibody (1/1000 dilution) (66 (link)) and mouse anti-HA mAb (HA-7; 1/400 dilution; Merck) as primary antibodies and Alexa Fluor 488-conjugated anti-rabbit IgG antibody and Alexa Fluor 594-conjugated anti-mouse IgG antibody (1/200 dilution each; Thermo Fisher Scientific) as secondary antibodies. Cells were mounted on glass microscope slides with ProLong Gold Antifade reagent (Thermo Fisher Scientific) and observed under a Leica DM5000B microscope (Leica Microsystems).

At passage number 6, hWJ-MSCs were visualized with bright field microscopy followed by flow cytometric analysis of cell surface markers. The cells were stained with fluorescein isothiocyanate-conjugated mouse monoclonal antibodies against human cluster of differentiation (CD) 105 (AbD Serotec, Oxford, UK), CD90 (Acris Antibodies, San Diego, CA, USA), CD73 (BD Biosciences, Franklin Lakes, NJ, USA), CD45 (BD Biosciences), CD34 (BD Biosciences), CD19 (Millipore, Billerica, MA, USA) and CD14 (Millipore), and human leukocyte antigen–antigen D related (HLA-DR) (BD Biosciences). An Alexa-Fluor 594-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific) was used to detect the unconjugated mouse monoclonal antibodies against human CD73 (BD Biosciences) and CD19 (Millipore). Antibodies were diluted in 1% FBS and phosphate-buffered saline (PBS) to their working concentration and incubated with hWJ-MSC for 15 min at 4 °C. At least 10′000 events were acquired on a LSR II flow cytometer (BD Biosciences) and data were analyzed using the FlowJo software (Tree Star, Inc., Ashland, OR, USA).

WJ-MSC were analysed by flow cytometry for cell surface markers as previously described [7 ]. The cells were stained with an adenomatous polyposis coli protein-conjugated (APC) mouse monoclonal antibody against CD105 (562408, BD Biosciences Inc.) and FITC-conjugated mouse monoclonal antibodies against CD90 (SM1170F, OriGene Technologies, Inc., Rockville, MD, USA), CD45 (555482, BD Biosciences Inc.), CD34 (555821, BD Biosciences Inc.), CD14 (MAB1219F, Merck KGaA), and human leukocyte antigen–antigen D related (HLA-DR) (555811, BD Biosciences Inc.), as well as the unconjugated antibodies against CD73 (550256, BD Biosciences Inc.) and CD19 (FCMAB184F, clone HD37, Merck KGaA). An Alexa-Fluor 594-conjugated anti-mouse IgG antibody (a11005, Thermo Fisher Scientific Inc.) was used to detect CD73 and CD19 antibodies. All antibodies were diluted in PBS containing 1% FBS to their respective working concentrations (Supplementary Table 1) and incubated with WJ-MSC for 15 min at 4 °C. At least 10’000 events were acquired on a LSR II flow cytometer (BD Biosciences Inc.) and data were analysed using FlowJo™ v10.8 Software (BD Biosciences Inc.).

IECs from Mettl3ΔIEC mice as well as from the wild-type littermate control mice were isolated as described in previous section. Isolated IEC were centrifuged onto glass slides and fixed with 4% Paraformaldehyde for 30 min at room temperature. Subsequently, permeabilized and blocked with PBS containing 0.1% Triton-X-100% and 5% bovine serum albumin for 1 hr at room temperature. Double-stranded RNA (dsRNA) was labeled by a mouse monoclonal antibody J2 (Scisons) for 2 hr at room temperature, followed by incubation with anti-mouse IgG Alexa Fluor 594-conjugated antibody (Invitrogen) for 1 hr, and cells nuclei were visualized with 4,6-diamidino-2-phenylindole (DAPI, Invitrogen). All fluorescence images were analyzed via confocal imaging using Zeiss LSM880.