Alexa fluor 488 conjugate

Cells grown on coverslips were fixed with using 3.7% formaldehyde in phosphate buffered saline (PBS) for 10 min. Cells were then permeabilized in 0.1% Triton X-100 in PBS, and washed in 0.5% Bovine Serum Albumin in PBS. Detection was performed using a rabbit polyclonal Runx1 antibody (Cell Signaling Technology #4336), a mouse monoclonal Vimentin (Santa Cruz Biotechnology sc-6260), a rabbit polyclonal N-cadherin (Santa Cruz Biotechnology sc-7939) and a mouse monoclonal to E-cadherin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Staining was performed using fluorescent secondary antibodies; for rabbit polyclonal antibodies a goat anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor® 488 conjugate (Life Technologies A-11008), was used and for mouse monoclonal a F(ab')2-goat anti-mouse IgG (H+L) secondary antibody, Alexa Fluor® 488 conjugate was used (Life Technologies A-11001).

Single-cell suspensions of hiPSCs were prepared by Accutase®-treatment. All steps were performed at 4 °C. In total, 2 × 10⁵ cells were stained per sample. Centrifugation steps were performed at 250 g and 4 °C. Cells were washed two times in 1X BD Perm/Wash™ buffer (BD Biosciences) prior to 20 min of incubation in PERM/FIX (BD Biosciences) solution for permeabilization and fixation of cells followed by another washing step and primary antibody incubation. For analysis of Oct4, Sox2 and Nanog, cells were incubated for 30 min at 4 °C with PE mouse anti-Oct3/4 (1:50; Human Isoform A, BD Biosciences), rat anti-Sox2 (1:50; Alexa Fluor® 488 Conjugate, eBioscience, USA) and Nanog (D73G4) XP® Rabbit mAb (1:50; Alexa Fluor® 647 Conjugate, Cell Signaling Technology, USA). Corresponding isotype controls were incubated with PE mouse IgG1 kappa isotype control (1:50; BD Biosciences), rat IgG2a kappa isotype control Alexa Fluor® 488 Conjugate (1:50; eBioscience) and Rabbit (DA1E) mAb IgG XP® Alexa Fluor® Conjugate 647 (1:100; Cell Signaling Technology). Afterwards, cells were washed two times in 1X BD Perm/Wash™ buffer followed by one washing step in FACS buffer (PBS⁻, 1% FCS, 2 mM EDTA (Sigma-Aldrich). Cells were analysed using a BD FACS Accuri C6 plus flow cytometer (BD Biosciences).

CHO-TRPA1 cells were seeded in glass coverslips and exposed to LPS from E. coli Alexa Fluor™ 488 Conjugate or S. minnesota Alexa Fluor™ 488 Conjugate (ThermoFisher Scientific, Waltham, MA, USA) during 10 min and CellMask™ Deep Red Plasma Membrane Stain (ThermoFisher Scientific) during 2 min. After treatment, cells were washed and fixed with cold paraformaldehyde and mounted in glass slides using DAPI-containing mounting solution (VectaShield, Vector Laboratories, Burlingame, CA, USA). The confocal images of labeled cells were collected using the optimal pinhole size for the 63X oil objective of a Zeiss LSM 880 Airyscan microscope (Carl Zeiss AG, Oberkochen, Germany). For quantification of cell images we analyzed a minimum of 10 cells from different slides per condition using the ImageJ software^{59 (link)}.

Tissue sections or primary cells were fixed and blocked with 5% Donkey serum (Sigma-Aldrich, USA). Samples were then incubated with primary antibodies at 4°C overnight. The following antibodies were used for staining: Bglap (Abcam, USA, ab93876, 5 ug/ml); Tppp3 (Abcam, USA, ab150998, 1:50); Mkx (Lifespan Biosciences Inc., USA, LS-B8063, 1 ug/ml); Gli1 (R&D, USA, MAB3324, 10 ug/ml); Gli2 (R&D, USA, AF3635, 5 ug/ml); pH3 (Santa Cruz, USA, sc-8656-R, 1:200); Donkey anti-rabbit IgG (H + L), Alexa Fluor 594 conjugate (Thermo Fisher Scientific, USA, A21207, 1:500); Donkey anti-rat IgG (H + L), Alexa Fluor 488 conjugate (Thermo Fisher Scientific, USA, A21208, 1:500); Donkey anti-goat IgG (H + L), Alexa Fluor 488 conjugate (Thermo Fisher Scientific, USA, A11055, 1:500); and Goat anti-rabbit IgG (H + L), Alexa Fluor 633 conjugate (Thermo Fisher Scientific, USA, A21072, 1:500). Samples were washed with PBS and incubated with secondary antibodies at room temperature for 1 hr. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific, USA). Signals were detected under fluorescence microscopy. For TUNEL assay, procedures were followed as described by the manufacturer using the ApopTag Plus In Situ Apoptosis Fluorescein Detection Kit (Millipore, USA, s7111).

Cells were fixed with ice-cold 4% paraformaldehyde (Biosesang, Gyeonggi, Korea) for 20 minutes at room temperature (RT) and permeabilized with 1% Triton X-100 (Sigma) for 5 minutes at RT. After washing with PBS, the cells were treated in a blocking solution containing PBS supplemented with 0.1% Triton X-100 (Sigma), 5% normal goat serum (Vector, Olean, NY, USA), and 5% normal horse serum (Vector) for 1 hour at RT. Then, the cells were incubated with primary antibodies overnight at 4°C; Nestin (1:2,500, Novus Biologicals, Littleton, CO, USA), STEM121™ (1:500, StemCells, Inc.), MAP-2 (1:200, Santa Cruz, Dallas, TX, USA), GFAP (1:2,000, Abcam). Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate (1:500, Thermo Fisher Scientific), Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate (1:500, Thermo Fisher Scientific), and Goat anti-Chicken IgG (H+L) Secondary Antibody [DyLight 488] (1:500, Novus Biologicals) were used as secondary antibodies for 2 hours at RT. The cells were treated with DAPI (1:1,000, Thermo Fisher Scientific) for 5 minutes at RT. After washing with PBS, the slides were mounted with Vectashield™ (Vector) and analyzed using an Axio observer Z1 as a fluorescence microscope (Zeiss, Jena, Germany).