The T cells were labelled with 5 μM carboxyfluorescein succinimidyl ester (CFSE; Sigma–Aldrich, USA) according to the manufacturer’s instructions, and then cocultured with DC, Rapa-DC or CsA-DC. After 5 days of coculture, the cells were harvested and labelled with the following fluorochrome-conjugated monoclonal antibodies: CD3-APC (clone: OKT3, eBioscience, USA), CD69-FITC (clone: FN50, eBioscience, USA) and CD25-PE (clone: BC96, BD Pharmingen, USA) to identify T cells or CD11c-APC and PD-L1-FITC (clone: MIH1, BD Pharmingen, USA) to identify DCs. All procedures were performed according to the manufacturers’ protocols. The cells were prepared for flow cytometry analysis and analysed as described above.
Pd l1 fitc
Manufactured by BD
Sourced in United States
PD-L1-FITC is a fluorescently labeled antibody that binds to the Programmed Death-Ligand 1 (PD-L1) protein. PD-L1 is a cell surface protein that plays a role in the regulation of the immune response. The FITC label allows for the detection and quantification of PD-L1 expression on cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
Pd l2 pe, by BD (2 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Cd69 fitc, by Thermo Fisher Scientific (1 mentions)
Cd3 apc, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Merck Group (1 mentions)
Cd11c apc, by BD (1 mentions)
Lsr flow cytometer, by BD (1 mentions)
Igg1 pe, by BD (1 mentions)
Igg1 fitc, by BD (1 mentions)
Pd 1 pe, by BD (1 mentions)
Ctla 4 pe, by BD (1 mentions)
Cd4 pc5, by BD (1 mentions)
Fc500, by Beckman Coulter (1 mentions)
Cd80 fitc, by BD (1 mentions)
Cd83 pe, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Cd86 pe, by BD (1 mentions)
Facs buffer, by BD (1 mentions)
Cd14 percp, by BD (1 mentions)
Tlr2 fitc, by Thermo Fisher Scientific (1 mentions)
6 protocols using pd l1 fitc
1
T Cell Activation Assay with DCs
2
Evaluating T-cell Exhaustion in T-ALL
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To recapitulate chronic antigen stimulation in vitro, freshly isolated PBMCs derived from healthy donors were co-cultured at 10:1 E:T ratio, as previously described for cytotoxic assay, with Cell Trace Far Red labeled CD1a+ T-ALL cells in the presence of CD1a x CD3ε alone, nivolumab alone, avelumab alone, combination of CD1a x CD3ε + nivolumab and CD1a x CD3ε + avelumab, for 5 days. Cell Trace Violet labeled CD1a+ T-ALL cells and drugs were then added to the culture. After 5 days, T cell exhaustion markers were evaluated by multiparametric flow cytometry panel (CD4-FITC, CD8-APC-H7, CD69-PE, PD-1-PE-CF594, TIM3-BV421, TIGIT-BV711, PD-L1 FITC BD Biosciences) and cytotoxicity was assessed by flow cytometry (ATTUNE NxT, Thermo Fisher Scientific).
3
Evaluating THLE-2 cell immune markers
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The detailed procedures were previously described [56 (link)]. THLE-2 cells (2 × 105) were treated with 100 µg/mL IPIL or 3:1 ratio of THLE-2 cell culture medium:T-CM. Twenty-four hours after the treatment, THLE-2 cells were trypsinized, pelleted, and washed 2× with 1× FACS buffer (1 × PBS + 1% FBS). The THLE-2 cells were then stained with the following antibodies including IgG isotype controls: PD-L1 FITC (BD Bioscience, cat# BD558065, San Jose, CA, USA), mouse IgG1k FITC (BD Bioscience, cat# 555748), CTLA-4 PE (Abcam, cat# ab210254), and mouse IgG2a PE (Abcam, cat# ab91363). Samples were analyzed with BD LSR Flow cytometer using 488, 575 filters. The data were analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA). Flow cytometry data shown in the figures of this manuscript are representative of at least two independent experiments. Each cell treatment was coming from a single well.
4
Phenotyping Cytotoxic T Cells via Flow Cytometry
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The antibodies used for CTL phenotyping (anti-CD3-ECD, CD4-PC5, CD8-PC7, PD-1-PE, CTLA-4PE, PD-L1 FITC; IgG1-FITC, IgG1-PE) were all obtained from BD Biosciences. To investigate competitive binding of staining antibody with blocking PD-1 mAb, flow cytometric analysis was performed using two different clones of staining antibody after 48 hrs: primary PD-1 (MIH4), secondary IgG1 FITC and PD-1 conjugated with PE.
Positive staining for PD-1 and PD-L1 was determined by comparing with the respective isotype control. The cell suspensions were incubated with mAb for 30 min at 4°C and then washed twice in PBS. PD-1 mAb-treated co-cultures were incubated with an anti-CD107a antibody and degranulation assay was performed.29 (link) The cells were acquired on an FC 500 (Beckman Coulter) and analyzed by the CXP analysis software.
Positive staining for PD-1 and PD-L1 was determined by comparing with the respective isotype control. The cell suspensions were incubated with mAb for 30 min at 4°C and then washed twice in PBS. PD-1 mAb-treated co-cultures were incubated with an anti-CD107a antibody and degranulation assay was performed.29 (link) The cells were acquired on an FC 500 (Beckman Coulter) and analyzed by the CXP analysis software.
5
Phenotypic Characterization of CD1c+ DCs
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Cells were incubated with appropriate monoclonal anti bodies: CD11c-APCw (BD Pharmingen), CD1c-FITC (eBioscience), CD83-PE (BD Pharmingen), CD80-FITC (BD Pharmingen), CD86-PE (BD Pharmingen), CD40-PE (BD Pharmingen), PD-L1-FITC (BD Pharmingen), PD-L2-PE (BD Pharmingen), HLA-DR-PE (BD Pharmingen), CCR7-APC-e Fluor (eBioscience), TLR2-FITC (eBioscience), TLR4-PE (eBioscience), CD64-FITC (BD Pharmingen), and DEC-205-PE (BD Pharmingen). In addition, CD14-PerCP (BD Pharmingen) monoclonal antibody was used to confirm the differentiation of DCs. Isotype controls were cells stained with IgG1 conjugated with the respective fluorochromes (BD Pharmingen, eBioscience). After 30 min of incubation at 4°C, cells were washed twice in FACS buffer (Becton Dickinson). The expression of individual markers on gated CD1c+ DCs was determined by flow cytometry (FACSCalibur, Becton Dickinson) and analyzed by Cell Quest software (Becton Dickinson). The results were based on analysis of at least 100,000 cells and were shown as the percentage of positively labeled cells. In addition, the mean channel fluorescence values of gated DCs positive for individual markers were determined.
6
Immunotherapy and FLOT Regimen in OAC
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OE33 cells and SK-GT-4 cells were treated with nivolumab (10 μg/ml, Opdivo, Bristol-Myers Squibb, USA), atezolizumab (10 μg/ml, Tecentriq, Roche, USA), A2aR antagonist (3 μM, ZM 241385 catalogue # 1036/10 Bio-techne, USA) or a MEK inhibitor (0.001 μM, U0126 Merck, USA) in the absence or presence of FLOT chemotherapy regimen. Doses of the FLOT chemotherapy regimen were pre-optimised to reduce the viability of cells by 50% (IC50 dose) as previously described (OE33 cells: 0.8249 µM 5-FU, 2 µM oxaliplatin and 0.001 µM of docetaxel and SK-GT-4 cells: 50 µM 5-FU, 10 µM oxaliplatin and 0.01 µM docetaxel) or cells were treated with the vehicle control (0.002% DMSO, 0.2% H2O)35 . Trypsinised OE33 cells or SK-GT-4 cells were stained with Zombie Aqua™, Violet™ or NIR™ viability dye (BioLegend, USA). Antibodies used for staining included LAG-3-FITC, CD160-PerCPCy5.5, PD-1-PE/Cy7, TIGIT-PE/Cy7 (BioLegend, USA), TIM-3-AF647, PD-L1-FITC, PD-L2-PE (BD Bioscience, USA) and A2aR-PE (Bio-techne, USA). OAC cells were resuspended in FACS buffer and acquired using BD FACS CANTO II (BD Biosciences, USA) using Diva software and analysed using FlowJo v10 software (TreeStar Inc.).
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