Blood dendritic cells antigen-3 DC activation status was assessed using surface stain markers CD40, CD80, CD86, and CCR7 from BD Biosciences. Cells were stained with fluorescent antibodies for 30 min on ice, washed twice with BD FACS staining buffer (DPBS contains 2% FBS and 0.09% sodium azide) and then acquired on a BD LSR II. Mean fluorescence intensity and cell percentages were determined by FlowJo V9.7 (Treestar). Cytokine production by day 7 primed T cells was assessed by intracellular cytokine staining (ICS) after initial staining with Cell Trace violet to detect proliferation (Life Technologies). Day 7 primed T cells were stimulated with PMA (50 ng/mL; EMD Millipore) and Ionomycin (1 μM; Sigma, St. Louis, MO, USA) in the presence of Golgi Stop (1 μl/mL; BD Bioscience) for 6 h. After incubation, cells were first surface stained with antibodies to CD25 (BD) and HLA-G (BioLegend), 30 min on ice. Next, cells were cultured using Live/Dead stain (Life Technologies) according to manufacturer’s protocol. Finally, cells were stained intracellularly with antibodies against IL-4 (eBiosciences) and IFN-γ (BD). Data were collected using BD LSRII and analysis was performed with FlowJo Software V9.7 (Treestar).
Hla g
Manufactured by BioLegend
Sourced in United States
HLA-G is a lab equipment product that functions as a human leukocyte antigen (HLA) class Ib molecule. It plays a role in immune regulation and is involved in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Hla dr, by BioLegend (2 mentions)
Ionomycin, by BioLegend (2 mentions)
Clone 87g, by BioLegend (2 mentions)
Anti hla c, by BioLegend (2 mentions)
Clone w6 32, by BioLegend (2 mentions)
Brefeldin a, by BioLegend (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Facs staining buffer, by BD (1 mentions)
Live dead stain, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Lsr 2, by BD (1 mentions)
Golgistop, by BD (1 mentions)
Ifn γ, by BD (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Zoledronic acid, by Merck Group (1 mentions)
Tcrαβ, by Thermo Fisher Scientific (1 mentions)
Phospho stat2 tyr690, by Cell Signaling Technology (1 mentions)
Hla g, by Cell Signaling Technology (1 mentions)
Alexa fluor 488, by BioLegend (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
7 protocols using hla g
1
Evaluating Dendritic Cell Activation and T-Cell Cytokine Production
2
Immunophenotyping of Immune Cells
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Zoledronic acid, LPS (lipopolysaccharide), puromycin, and G418 were obtained from Sigma‐Aldrich. Antibodies against human HLA‐G (sc‐21799) and β‐actin (sc‐47778) were purchased from Santa Cruz Biotechnology. PD‐L1 antibody (17952‐1‐AP) and PD‐L1 (22C3) were purchased from Proteintech and Agilent Technologies, respectively. Antibodies specific for CD3ε (NB600‐1441), PD‐L1 (FAB1561R, FAB1561G), CD4 (FAB3791R), and CD8 (NBP2‐34590AF488) were purchased from Novus Biologicals. HLA‐G (PE: #335906, Alexa Fluor 488: #335918), PD‐1 (#367412), CD3 (#317318), CD8 (#344704), CD56 (#304611), CD66b (#305104), and Vδ2 (#331418) were purchased from BioLegend. Anti‐Tyk2 (phospho‐Tyr1054/1055) (orb505746) antibody was obtained from Biorbyt. Antibodies specific for phospho‐ZAP70/Syk (Tyr319, Tyr352) (#MA5‐36963) and HLA‐G (MEM‐G/2: #MA1‐19394, 87G: MA1‐10356) were purchased from Thermo Fisher Scientific. Antibodies for CD14 (#12‐0149‐42), TCRγδ (#12‐9959‐42), TCRαβ (#11‐9955‐42), and NKG2D (#12‐5878‐42) were purchased from eBioscience. Vγ9 (#555732), hMito (ab92824), and VHH (iFluor 647: A02019, HRP: A2016) were purchased from BD Pharmingen, Abcam, and GenScript, respectively. Phospho‐Stat2 (Tyr690) (#77366) and HLA‐G (#79769) were obtained from Cell Signaling Technology. Recombinant human interleukin 2 (IL‐2) was obtained from Thermo Fisher Scientific.
3
Immunophenotyping of Immune Cells
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Cells were washed with PBS supplemented with 2% FBS/PBS and FITC-, APC- and PE-conjugated mAbs directed to CD1a, CD14, CD86, HLA-DR, CD83, CD4, CD25 (BD Biosciences), ILT-2/CD85j, and HLA-G (BioLegend, San Diego, CA, United States) or the corresponding isotype controls were added at saturating concentrations for 30 min at 4°C. Then, two additional washes were performed, and cells were fixed with 1% paraformaldehyde. Stained cells were acquired using an FACS Calibur and FACSAria II cytometers and results were analyzed using FlowJo 7.6 Software.
4
Immunophenotyping of Extracellular Vesicles
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Anti‐CD63 magnetic beads (Dynabeads 10606D, Thermo Fisher Scientific; 15 μl containing 1.5 × 105 beads) were incubated with samples containing 107–108 EV particles in a volume of 50 μl bovine serum albumin (BSA), 1 mg/ml in PBS (BSA‐PBS) overnight at 4°C with gentle shaking. Preliminary experiments indicated that these conditions captured >85% of EV particles measured by TRPS or fluorescent‐labeled EVs (data not shown). The samples were rinsed 3× in PBS by magnetic capture and then incubated in a volume of 50 μl PBS‐BSA containing fluorescent antibodies to one or combinations of the following markers: CD81, CD9, CD29, CD13, CD90, CD36, HLAG, HLA‐DR, CD34, CD146, CD107a, CD274, CD166, CD44, CD74, or CD59 (BioLegend, CA) for 2 hours at room temperature. After 3× rinsing in PBS the beads were analyzed by flow cytometry using a BD FACS Aria III instrument. A majority of beads with mean fluorescence greater than that of the isotype control antibody was considered indication of presence of the targeted protein (Supporting Information Fig. S1). Triplicate analyses were carried out for all samples.
5
Trophoblast-Decidual Cell Interactions
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Freshly isolated Tros were seeded at a density of 2 × 105 cells/ml per well in Matrigel (Coring, U.S.A)-coated 24-well plates overnight. The cells were then washed twice with phosphate-buffered saline (PBS, HyClone, U.S.A). Equal numbers of dCD14+ cells or pCD14+ cells were added to each well. In some wells, anti-HLA-C (10 μg/ml, clone W6/32; Biolegend, U.S.A), HLA-G (10 μg/ml, clone 87 G; Biolegend, U.S.A) were added. dCD14+ cells were also cultured with HTR8/Svneo cells (ATCC, www.atcc.org ) or DSCs for 48 h. In some wells, dCD14+ cells (2 × 105 cells) were plated in the upper chamber (0.4 mm pore size cell culture inserts, Millipore, Germany), while Tros were plated in the lower chamber to establish indirect cell contact.
Freshly isolated dCD4+ T cells co-cultured with Tim-3+dMφs or Tim-3−dMφs at a 1: 1 ratio. In some experiments, Tim-3+dMφs were pretreated with anti-Tim-3 (10 μg/ml, clone F38-2E2, BioLegend, U.S.A.), or anti-CD132 (10 μg/ml, clone TUGh4, BioLegend, U.S.A.), or anti-IL-4 (10 μg/ml, clone F38-2E2, BioLegend, U.S.A.). Phorbol 12-myrstate 13-acetate (PMA) (50 ng/ml, Biolegend, U.S.A.), ionomycin (1 μg/ml, Biolegend, U.S.A.) and brefeldin A (10 mg/ml, BioLegend, U.S.A.), were added 4 h before the end of the 48 h co-culture. The supernatants were then collected for intracellular cytokine analysis of T cells.
Freshly isolated dCD4+ T cells co-cultured with Tim-3+dMφs or Tim-3−dMφs at a 1: 1 ratio. In some experiments, Tim-3+dMφs were pretreated with anti-Tim-3 (10 μg/ml, clone F38-2E2, BioLegend, U.S.A.), or anti-CD132 (10 μg/ml, clone TUGh4, BioLegend, U.S.A.), or anti-IL-4 (10 μg/ml, clone F38-2E2, BioLegend, U.S.A.). Phorbol 12-myrstate 13-acetate (PMA) (50 ng/ml, Biolegend, U.S.A.), ionomycin (1 μg/ml, Biolegend, U.S.A.) and brefeldin A (10 mg/ml, BioLegend, U.S.A.), were added 4 h before the end of the 48 h co-culture. The supernatants were then collected for intracellular cytokine analysis of T cells.
6
Characterizing hAECs Purity and Plasticity
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To evaluate the characteristics and purity of hAECs, we employed flow cytometry to detect the expression of relative cell markers. E-cadherin (Biolegend, San Diego, CA, USA) and SSEA4 (Biolegend, San Diego, CA, USA) were used to test the epithelial properties and plasticity of hAECs, respectively. CD34 (Invitrogen, Grand Island, NY, USA), CD45 (Biolegend, San Diego, CA, USA), CD31 (Invitrogen, Grand Island, NY, USA), CD144 (Invitrogen, Grand Island, NY, USA), CD73 (Invitrogen, Grand Island, NY, USA) and CD90 (Invitrogen, Grand Island, NY, USA) were used to evaluate the purity and basic characteristics of hAECs. To indicate the immunogenicity of hAECs before and after inducing treatment, HLA-DR (Biolegend, San Diego, CA, USA), HLA-DQ (Biolegend, San Diego, CA, USA) and HLA-G (Biolegend, San Diego, CA, USA) were stained after cells incubated with 10 ng/mL IFN-γ (Peprotech, Cranbury, NJ, USA) for 3 days. All procedures followed the manufacturers’ instructions, and later, analysis was conducted with flow cytometry (Beckman, Brea, CA, USA). Analyses were performed on five biological replicates.
7
Trophoblast-CD8+ T Cell Interaction
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Freshly isolated trophoblasts were seeded at a density of 2 × 105 cells/ml per well in Matrigel (Coring, USA)-coated 24-well plates overnight. The cells were then washed twice with phosphate-buffered saline (PBS, HyClone, USA). Equal numbers of dCD8+ T cells or pCD8+ T cells were added to each well. In some wells, anti-HLA-C (10 μg/ml, clone W6/32; Biolegend, U.S.A), HLA-G (10 μg/ml, clone 87G; Biolegend, USA) were added. dCD8+T cells were also cultured with plate-bound anti-CD3 antibody (OKT-3; 5 μg/ml, Biolegend, USA) plus soluble anti-CD28 antibody (28.2; 1 μg/ml, Biolegend, USA), or HTR8/Svneo cells or DSCs for 48 h. In some wells, dCD8+ T cells (2 × 105 cells) were plated in the upper chamber (0.4 mm pore size cell culture inserts, Millipore, Germany), while trophoblasts were plated in the lower chamber to establish indirect cell contact. Phorbol 12-myrstate 13-acetate (PMA) (50 ng/ml, Biolegend, USA), ionomycin (1 μg/ml, Biolegend, USA) and brefeldin A (10 mg/ml, BioLegend, USA), were added 4 h before the end of the 48 h culture for intracellular cytokine analysis. The cells were then harvested for flow cytometry analysis.
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