Fitc labeling kit nh2

Subsets analysis was performed as described in our previous study [20 (link)]. Briefly, the cells were washed in PBS containing 0.05% sodium azide and 0.5% bovine serum albumin (BSA-PBS). These cells (1 × 10⁵ to 1 × 10⁶) were incubated with fluorescence labeled monoclonal antibodies (mAbs) as below for 1hr at 4 °C. After washing with BSA-PBS, the cells were suspended in BSA-PBS containing propidium iodide (1 µg/mL, Sigma Aldrich). The relative fluorescence intensities were examined by multicolor flowcytometry (FACS Canto^TM II system, Becton Dickinson, Franklin Lakes, NJ, USA). Fluorescence labeled anti-MHC class II (×200 dilution, TH81A5, Monoclonal Antibody Center at Washington State University, Pullman, WA, USA), anti-IgM (×100 dilution, PIG45A2, Monoclonal Antibody Center at Washington State University), anti-CD4 (×200 dilution, PT90A, Monoclonal Antibody Center at Washington State University), anti-CD8 (×200 dilution, PT36B, Monoclonal Antibody Center at Washington State University) and anti-γδ (×100 dilution, PGBL22A, Monoclonal Antibody Center at Washington State University) were used. For fluorescent labeling, HiLyte^TM Fluor 647 labeling kit-NH₂ (Dojindo Laboratories, Kumamoto, Japan), HiLyte^TM Fluor 555 labeling kit-NH₂ (Dojindo Laboratories) and FITC labeling kit-NH₂ (Dojindo Laboratories) were used according to manufacturer’s instruction.

Immunocytochemical analysis was performed as described previously [16 (link)–18 (link)]. The following primary antibodies were used in this study: anti-SSEA4 (1:300 dilution; Millipore), anti-TRA-1-60 (1:300 dilution; Millipore), anti-TRA-1-81 (1:300 dilution; Millipore), anti-Oct-3/4 (1:300 dilution; Santa Cruz Biotechnology), anti-human Nanog (1:800 dilution; Cell Signaling Technology), anti-Class IIIβ-tubulin (TUJ1; 1:500; Covance), anti-human smooth muscle actin (SMA; 1:50; DAKO), and anti-α-fetoprotein (1:100; R&D systems). Cells were incubated with a primary antibody diluted in 1% bovine serum albumin (BSA) and 5% serum containing PBS at 4°C overnight. Secondary staining was performed with an appropriate secondary antibody-conjugated to Alexa Fluor 488 or Alexa Fluor 594 (1:300; Life technologies) for 1 h at room temperature. Lectin staining was performed as described previously [19 (link),20 (link)]. Briefly, rBC2LCN (Wako) was fluorescent-labeled using FITC Labeling Kit-NH2 (Dojindo) according to the manufacturer’s instruction. Next, 10 μg/mL of FITC-conjugated rBC2LCN in 1% BSA containing PBS was used for staining of 4% paraformaldehyde-fixed cells for 1 h at room temperature. Cells were counterstained with DAPI (Dojindo). Images were collected with a BIOREVO BZ-9000 fluorescence microscope (Keyence).