Single-cell suspensions were initially incubated with an Fc receptor blocking antibody (Biolegend, San Diego, CA, USA) and stained with the following antibodies for surface analysis: anti-human CD3 (SK7, BD Biosciences), anti-human CD4 (RPA-T4, BD Biosciences), anti-human CD25 (M-A251, BD Biosciences), anti-human CD127 (HIL-7R-M21, BD Biosciences), anti-human CXCR3 (1C6, BD Biosciences), anti-human CCR4 (L291H4, Biolegend), and anti-human CCR6 (G034E3, Biolegend). PBMCs were stained with anti-CD3 (SK7, BD Biosciences), anti-CD4 (RPA-T4, BD Biosciences), anti-CD25 (M-A251, BD Biosciences) prior to fixation, permeabilization and staining for Foxp3 (259D, Biolegend). For analysis of ST2+ Tregs, PBMCs were stained for CD4 and ST2 (RMST2-33, Invitrogen, Carlsbad, CA, USA) prior to staining for Foxp3. For analysis of α-smooth muscle actin (α-SMA)+ OFs after coculture with Tregs, OFs were fixed and permeabilized after harvest and stained with an anti-α-SMA antibody (1A4/asm-1, Novus Biologics, Minneapolis, USA).
Rpa t4
Manufactured by BD
Sourced in United States, United Kingdom
The RPA-T4 is a laboratory equipment designed to automate repetitive pipetting tasks. It features a compact size and user-friendly interface, allowing for accurate and efficient liquid handling.
Automatically generated - may contain errors
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62 protocols using rpa t4
1
Characterization of Regulatory T Cells
2
Multiparameter Flow Cytometry Analysis
3
Characterization of Anti-CD4 Monoclonal Antibodies
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The following anti-human CD4 mAbs were used: BT-061 (Biotest AG, Dreieich, Germany), OKT-3 (BioLegend, San Diego, CA, USA), RPA-T4 (BD Pharmingen, San Jose, CA, USA), SK3 (BioLegend), MT310 (Santa Cruz Biotechnology, Dallas, TX, USA), QS4120 (Abnova, Neihu District, Taipei City, Taiwan), B-A1 (Acris Antibodies, San Diego, CA, USA), EDU-2 (Santa Cruz Biotechnology), MT441 (Ancell Corporation, Bayport, MN, USA), OKT-4 (BioLegend).
4
Quantifying CTLA-4 Expression in Activated CD4+ T Cells
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Primary human CD4+ T cells were purified from whole PBMCs using EasySep™ Human CD4+ T Cell Enrichment Kit (Stemcell Tech.) and activated with CD3/CD28 beads for 16 hours in the presence of CHO cells expressing CD80-GFP .. After 16 h CTLA-4 expression (anti-CTLA-4-PE, BNI3, BD bioscience) was assessed by staining cells at 37°C for the final 2 hours.. Subsequently cells were stained for CD4 (RPA-T4, BD bioscience, 1/50), , CD45RA (HI100, eBioscience, 1/200), CD127 (HIL-7R-M21, BD Bioscience, 1/50) and CD25 (2A3, BD bioscience. 1/50) then fixed and permeabilized and stained for FOXP3 (236A/E7, eBioscience, 1/50). Cells were gated on FOXP3+ cells and analyzed for GFP uptake.
5
Isolation of Human CD4+ CD25hi Tregs
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Human Treg (CD4+ CD25hi) were purified from PBMCs from healthy donors, after staining with the following antibodies at 1:100 dilution: FITC anti-human CD4 (BD PharMingen, clone RPA-T4, Cat.# 555346), PE anti-human CD25 (BD PharMingen, clone M-A251, Cat.# 555432). Treg cells were treated with Detach reagent (Invitrogen) to remove antibody. Treg cells were thus harvested and subjected to the following experiments.
6
Multiparametric Analysis of Tumor-Infiltrating T Cells
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Peripheral blood mononuclear cells were isolated from whole blood by Ficoll-Paque gradient centrifugation. Freshly resected tumor specimens were manually disrupted followed by digestion with collagenase IV (2.5 mg/mL) and DNase I (0.2 mg/mL) (Worthington Biochemical Corporation) for 1 hour. Tumor homogenates were separated on discontinuous 70%–30% Percoll (Sigma-Aldrich) gradients. Flow cytometric analysis was performed with antibodies targeting CD4 (BD Biosciences clone RPA-T4, V450 conjugated), CD8 (BD Biosciences clone RPA-T8, V500 conjugated), TIGIT (eBioscience clone MBSA43, PerCP-eFluor® 710 conjugated), CD226 (eBioscience clone TX25, FITC conjugated), and PD-1 (BD Biosciences clone EH12.1, Alexa Fluor® 647 conjugated). Cell viability was assessed using Live/Dead Cell Viability Assays (Life Technologies). Samples were run on a BD LSRFortessa or BD FACSAria II, as previously described. FlowJo software (Tree Star Inc.) was used for analysis after gating on live cells, with doublet exclusion followed by gating on CD4 and CD8 T cells.
7
Flow Cytometry Analysis of PBMCs and PFMCs
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PBMCs and PFMCs from TPE were obtained for flow cytometry as previously described (Cai et al., 2020 (link)). The samples were then stained with antibodies against CD3 (SK7), CD4 (RPA-T4), and CD8 (SK1; BD Biosciences) plus ghost dye (Tonbo Biosciences) for 30 min at 4°C. Following fixation and permeabilization for 30 min at room temperature, the cells were stained with mAbs against GZMB (GM26E7), GZMK (GB11), and GZMA (CB9; BD Biosciences) for 30 min at room temperature. All antibodies were validated by the manufacturer for flow cytometry application. The cells were resuspended in 200 µl of 2% paraformaldehyde, acquired using FACSDiva software (BD Biosciences), and analyzed using FlowJo software v10.
8
Plasma Cell Differentiation Assay
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B cells obtained from human PBMCs (described above) were seeded in 96-well plates at 2.5 × 106 cells/mL, then cultured at 37°C with 5% CO2 for 3 days in RPMI supplemented with 10% FBS, 100 U/mL Pen/Strep, 0.11 mg/mL sodium pyruvate, 10 mM HEPES, 2 mM glutamine, 55 μM 2ME with IL-21 (50 ng/mL), anti-CD40 (5 μg/mL, IC10 Southern Biotech), and R848 (Resiquimod, 3 μg/mL, Sigma-Aldrich). CAR T cells were added to B cells at a 1:1 cell ratio, keeping the total cell concentration between 0.5–1 × 106 in B cell media as above, but with the addition of IL-2 (50 ng/mL), IL-7 (5 ng/mL), and IL-15 (5 ng/mL). The relative proportion of CD4−CD8− BCMA+ plasma cells in each sample was then assessed 2 days post-addition of T cells by flow cytometry after staining with FITC anti-CD4 (RPA-T4, BD Bioscience), PerCP-Cy5.5 anti CD-8 (RPA-T8, BD Bioscience), PE anti-BCMA (19F2, BioLegend), Alexa700 anti-CD138 (MI15, Biolegend), and PE-Cy7 anti-CD19 (HIB19, eBioscience).
9
Comprehensive T-Cell Immunophenotyping by Flow Cytometry
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Cells harvested from overnight stimulation cultures were incubated with live/dead aqua V510 for 15 min on ice. The cells were then surface stained for 30 min on ice using anti-CD3-FITC (BioLegend, clone UCHT1, 1:200, Cat# 300406), anti-CD4-APC-H7 (BD Pharmingen™, clone RPA-T4, 1:200, Cat# 560158), and anti-CD8-PerCPCy5.5 (BD Biosciences, clone RPA-T8, 1:200, Cat# 560662) antibodies. After fixation and permeabilization with Cytofix and Perm (BD Biosciences, Cat# 554714) on ice for 15 min, intracellular staining was performed on ice for 30 min using anti-TFNα-PE-Cy7 (BD, clone MAb11, 1:200, Cat # 557647) and anti-IFNγ-APC (BD Pharmingen™, clone B27, 1:200, Cat# 554702) antibodies. After the final wash step, the cells were resuspended in 200 µL of FACS buffer. Samples were acquired using an FACSAria III instrument (BD Biosciences, San Diego, CA, USA) and analysed using the FlowJo software (Treestar, San Carlos, CA, USA).
10
Analysis of T Cell Subsets and Activation Markers
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Recombinant human TNF was purchased from Leinco Technologies (St Louis, MO, USA), and recombinant human IL-2 was purchased from Sigma-Aldrich (St Louis, MO, USA). Monoclonal antibodies against TNFR2 were produced in house or purchased from commercial vendors as previously described.8 (link) Fluorochrome-conjugated mAbs against human CD4 (RPA-T4), CD25 (M-A251), CD45RA (HI100, 2H2), CD45RO (UCHL1, 4HB), CD127 (hIL-7R-M21), HLA-DR (L243 (G46-6)) were purchased from BD Biosciences. Fluorochrome-conjugated monoclonal antibodies against CD4 (S3.5, Invitrogen, Carlsbad, CA, USA), CD120a (16803 R&D systems), CD120b (22235, R&D systems, Minneapolis, MN, USA), FOXP3 (259D, Biolegend, San Diego, CA, USA) were also used in this study.
Intracellular staining of FOXP3 and CD152 was performed using either FOXP3 Fix/Perm Buffer set (Biolegend) or Human FoxP3 Buffer set (BD Biosciences) according to the manufacturer's instructions. Flow cytometric data were obtained using FACSCalibur (BD Biosciences) flow cytometer. All the data were analyzed with Cellquest Software (BD Biosciences, San Jose, CA, USA).
Intracellular staining of FOXP3 and CD152 was performed using either FOXP3 Fix/Perm Buffer set (Biolegend) or Human FoxP3 Buffer set (BD Biosciences) according to the manufacturer's instructions. Flow cytometric data were obtained using FACSCalibur (BD Biosciences) flow cytometer. All the data were analyzed with Cellquest Software (BD Biosciences, San Jose, CA, USA).
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