Pe mouse anti human hla dr

Collected MSCs were incubated for 30 min at room temperature with the following specific antibodies: PE mouse anti-human CD29, FITC rat anti-human CD44, FITC mouse anti-human CD105, FITC rat anti-human CD45, APC mouse anti-human CD34, and PE mouse anti-human HLA-DR (all from BD Pharmingen). As a control, the cells were stained with the appropriate isotype antibodies. At the end of co-culture, the CD4⁺ T cell apoptosis was analyzed by using an Annexin V-PE Apoptosis Detection Kit I (BD Biosciences) according to the manufacturer’s instructions. To detect Treg cells, a Human Regulatory T Cell Staining Kit (eBioscience) containing an anti-CD4-FITC/CD25-APC cocktail and anti-Foxp3-PE was used according to the manufacturer’s instruction. In addition, we used a Human Th1/Th2/Th17 Phenotyping kit (BD Pharmingen) to analyze the T helper cell subsets. All samples were analyzed using a BD Biosciences Influx cell sorter.

The presence of HLA class I and II on the surface of spermatozoa was assessed by direct immunofluorescence using a BD FACSCalibur (BD Biosciences, USA) flow cytometer. Two tubes for each sample containing 1 × 10⁶ spermatozoa were prepared. One was incubated with phycoerythrin (PE) mouse anti‐human HLA‐ABC (clone: G46‐2.6, BD pharmingen, USA) and the other was incubated with phycoerythrin (PE) mouse anti‐human HLA‐DR (clone: G46‐6, BD pharmingen, USA) at room temperature for 30 min. After two washes with AllGrad Wash (400 g for 5 min), tubes were run through the flow cytometer. Data from at least 100,000 events were collected using forward scatter (a logarithmic amplifier) and side angle of light scatter (a logarithmic amplifier). As a negative control, we used unstained control. Isotype controls [Mouse IgG1, κ (clone: G46‐2.6, BD pharmingen, USA) and Mouse IgG2a, κ (clone: G46‐6, BD pharmingen, USA)] were used to determine background fluorescence (autofluorescence and non‐specific binding of antibodies). To remove background fluorescence, antibody titration was done and the optimal titer that showed minimum background was selected. Noteworthy, cell viability test was not performed because we removed abnormal and dead spermatozoa by AllGrad solution before staining. Fluorescence data were obtained with the logarithmic amplifier. We used flowJo vx software for data analyses.

H9‐derived MSCs and human bone marrow‐derived MSCs (Lonza, Walkersville, MD,
http://www.lonza.com) were grown to confluence, harvested by using 0.25% trypsin/EDTA, and resuspended in buffer containing phosphate‐buffered saline (PBS), 2% HEPES buffer, 2% FBS, and 0.1% bovine serum albumin (BSA) as previously described 43. Cells (1 × 10⁶) were incubated with phycoerythrin (PE) mouse anti‐human CD90, PE mouse anti‐human CD73, fluorescein isothiocyanate (FITC) mouse anti‐human CD44, FITC mouse anti‐human CD45, FITC mouse anti‐human HLA‐ABC, PE mouse anti‐human CD29, PE mouse anti‐human CD166, PE mouse anti‐human HLA‐DR, FITC mouse anti‐human CD105, or FITC mouse anti‐human CD31 (BD Biosciences, San Jose, CA,
http://www.bdbiosciences.com). Nonspecific fluorescence was determined by using isotype‐matched monoclonal antibodies. A total of 10,000 events were collected on a BD fluorescence‐activated cell sorting Calibur Flow Cytometer instrument by using CellQuest software (BD Biosciences). Analyses of results and corresponding graphs were generated by using FlowJo software (Tree Star, Ashland, OR,
http://www.flowjo.com) 434445.

Dental pulp tissues were isolated from extracted deciduous incisors (6–8-year-old donors). Parents of these donors signed written informed consent forms. Pulp tissues were isolated, washed, digested in 3 mg/ml collagenase type I (Sigma-Aldrich, United States) for 2 h and incubated with culture medium at 37°C in 5% CO₂. At 80% confluence, the cells were trypsinized and subcultured. The specific cell surface molecules were identified using flow cytometry. Briefly, cells at passage three were trypsinized and incubated with FITC mouse anti-human CD105 (Cat.561443), PE mouse anti-human CD34 (Cat.555822), PE mouse anti-human CD90 (Cat.555596), PE mouse anti-human HLA-DR (Cat. 555812) and BV510 mouse anti-human CD45(Cat.563204) (all from BD Biosciences, United States) on ice for 30 min. Then the cells were washed, resuspended and detected with a flow cytometry system (BD LSRFortessa, BD Biosciences, Franklin Lakes, NJ, United States).

Referencing a previous in vitro study [10 (link)], PBMCs were stained with a fluorescent dye and fluorescent conjugated antibodies: BD Horizon Fixable Viability Stain 780 (FVS780) viability dye, PE-Cy7 Mouse Anti-Human CD123 (BD, 560826), BB515 Mouse Anti-Human Neuropilin-1 (CD304) (BD, 566036), and APC Mouse Anti-Human CD86 (BD, 555660) following the manufacturer’s instructions, and then fixed with 4% paraformaldehyde. PBMCs were cultured at 1 × 10⁶ cells/mL in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 5% human AB serum (Sigma-Aldrich), 2 mM Glutamax (Thermo Fisher Scientific, Waltham, MA, USA), and 1% penicillin/streptomycin (Thermo Fisher Scientific), and stimulated in the presence of 10 µg/mL TLR-7/8 agonist R848 (InvivoGen, San Diego, CA, USA) for 4 h. Afterward, the cells were harvested and stained with FVS780, PE-Cy7 Mouse Anti-Human CD123, BB515 Mouse Anti-Human Neuropilin-1 (CD304), and PE Mouse Anti-Human HLA-DR (BD, 556644) as described above. Data were collected using CytoFLEX (Beckman Coulter, Brea, CA, USA) and analyzed using the FlowJo version 10 software (BD). Isotype controls (BD) were used to check the background caused by nonspecific antibody binding. pDCs were defined as CD123+CD304+ subsets, and CD86 and HLA-DR were used as activation markers for pDCs.