The isolated mononuclear lymphocytes from both the decidua and peripheral blood were directly stained for sorting. For FACS sorting, they were incubated with conjugated mouse anti−human antibodies, including anti-CD3 FITC (BioLegend, UK), anti-CD56 PerCp/Cy5.5 (BioLegend, UK), anti-CD4 BV421 (BD Biosciences, U.S.A.), and anti-CD8a PE/Cy7 (BioLegend, UK), for 30 min at 4 °C. After incubation, they were treated with LIVE/DEAD® Fixable Aqua Dead Cell Stain (Invitrogen Life Technologies, U.S.A.) for 10 min. CD3+ CD56−CD4−CD8+ cells were sorted on a BD FACS Aria-II machine to obtain a purity > 95%. For coculturing with either DSCs or trophoblast cells, CD8+T cells from the decidua and PBMC via magnetic affinity cell were sorted using CD8 MicroBeads (MiltenyiBiotec, Germany), according to the manufacturer’s instructions.
Anti cd4 bv421
Manufactured by BD
Sourced in United States
Anti-CD4-BV421 is a fluorescently-labeled monoclonal antibody that binds to the CD4 surface antigen. It is designed for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3 fitc, by BioLegend (2 mentions)
Anti cd8 bv711, by BioLegend (2 mentions)
Facsaria 2 machine, by BD (1 mentions)
Cd8 microbead, by Miltenyi Biotec (1 mentions)
Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (1 mentions)
Anti cd56 percp cy5, by BioLegend (1 mentions)
Anti cd8a pecy7, by BioLegend (1 mentions)
Golgiplug, by BD (1 mentions)
Anti ifn γ pe, by BD (1 mentions)
Anti cd8 fitc, by BD (1 mentions)
Macsquant flow cytometer, by Miltenyi Biotec (1 mentions)
Golgistop, by BD (1 mentions)
Rbc lysis buffer, by Merck Group (1 mentions)
Cell strainer, by SPL Life Sciences (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Live dead staining kit, by InvivoGen (1 mentions)
Anti il 5 apc, by BD (1 mentions)
Anti il 17a pe cy7, by BD (1 mentions)
Anti pd l1 pe, by Thermo Fisher Scientific (1 mentions)
Anti cd8 apc cy7, by BD (1 mentions)
8 protocols using anti cd4 bv421
1
Isolation and Sorting of CD8+ T Cells
2
Quantifying T-cell Cytokine Responses to Vaccine
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Two weeks after the final immunization, spleens from mice immunized with either the r-SG or f-SG vaccine were isolated and filtered through a cell strainer (70 µm; SPL Life Sciences, Pocheon, Republic of Korea). Red blood cells (RBCs) were lysed with RBC lysis buffer (Sigma-Aldrich) and washed with RPMI-1640 medium containing 10% fetal bovine serum (FBS; Biowest, Nuaillé, France). The cell suspension was seeded into a 48-well plate (2 × 106 cells/well) and stimulated with 10 µg/mL SG lysate, 0.5 µg/mL GolgiStop (BD Bioscience, San Diego, CA, USA), and 0.5 µg/mL GolgiPlug (BD Bioscience) at 37°C for 12 h. The cells were washed with PBS and stained with a Live/Dead Staining Kit (InvivoGen, San Diego, CA, USA), anti-CD8-FITC (BD Bioscience), and anti-CD4-BV421 (BD Biosciences) for 20 min at 23°C to stain T cell surface markers. Cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Bioscience) for 20 min at 4°C, and then intracellular cytokines were stained with anti-IFN-γ-PE (BD Biosciences), anti-IL-5-APC (BD Bioscience), and anti-IL-17A-PE-Cy7 (BD Bioscience) for 20 min at 23°C. After staining, the cells were analyzed using a MACS Quant flow cytometer (Miltenyi Biotec, San Diego, CA, USA) and FlowJo software (TreeStar, Ashland, OR, USA).
3
Isolation and Phenotyping of Human Neutrophils
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Peripheral blood mononuclear cells (PBMCs) were isolated via centrifugation on a Ficoll–Paque PLUS gradient (GE Healthcare, Uppsala, Sweden). Neutrophils were further isolated after dextran sedimentation and hypotonic lysis as previously described (20 (link)).
Fresh EDTA anti-coagulated peripheral blood (100 μl) was incubated with antibodies for 30 min in the dark. The following antibodies were used: Anti-CD66b-PE-cy7, anti-TLR2-FITC, anti-TLR4-APC, anti-CXCR1-APC, anti-CXCR2-FITC, anti-C5aR-APC, anti-CD64-PE, anti-CD177-APC, anti-CD62L-FITC, anti-CD11b-PE, anti-CD49d- FITC, anti-C5L2-PE, anti-CD3-BV421 and anti-PD-1-PE were obtained from Biolegend (San Diego, California, USA); anti-CD4-BV421, anti-CD8-APC-cy7, anti-CD38-FITC, and anti-HLA-DR-PerCP were purchased from BD Biosciences (Franklin Lakes, New Jersey, USA); anti-PD-L1-PE were bought from eBioscience (San Diego, California, USA). Isotype-matching antibodies were used as negative controls. After lysing the red blood cells, the remaining cells were washed and fixed for flow cytometry analysis.
Fresh EDTA anti-coagulated peripheral blood (100 μl) was incubated with antibodies for 30 min in the dark. The following antibodies were used: Anti-CD66b-PE-cy7, anti-TLR2-FITC, anti-TLR4-APC, anti-CXCR1-APC, anti-CXCR2-FITC, anti-C5aR-APC, anti-CD64-PE, anti-CD177-APC, anti-CD62L-FITC, anti-CD11b-PE, anti-CD49d- FITC, anti-C5L2-PE, anti-CD3-BV421 and anti-PD-1-PE were obtained from Biolegend (San Diego, California, USA); anti-CD4-BV421, anti-CD8-APC-cy7, anti-CD38-FITC, and anti-HLA-DR-PerCP were purchased from BD Biosciences (Franklin Lakes, New Jersey, USA); anti-PD-L1-PE were bought from eBioscience (San Diego, California, USA). Isotype-matching antibodies were used as negative controls. After lysing the red blood cells, the remaining cells were washed and fixed for flow cytometry analysis.
4
Profiling COVID-19 Patient Immune Responses
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Peripheral blood mononuclear cells (PBMCs) from COVID‐19 patients and healthy volunteers were separated by density gradient centrifugation using Pancoll (PAN Biotech, Aidenbach, Germany) and stored in liquid nitrogen until further processing. Cells were thawed and stimulated with 1 μg/ml of total Spike and Nucleocapsid antigen for 24 h. Subsequently, cells were stained with anti‐CD3‐fluorescein isothiocyanate (FITC), anti‐CD137‐phycoerythrin (PE), anti‐CD8‐Brilliant Violet 421 (BV421), anti‐CD4‐BV510 and 7‐aminoactinomycin D (7AAD) (all from BD Biosciences, San Jose, California, USA). Additionally, for COVID‐19 patients only, PBMC were stained ex vivo without prior peptide stimulation with anti‐HLA‐DR‐PE, anti‐CD3‐BV510, anti‐CD4‐BV421, anti‐CD8‐allophycocyanin‐cyanin 7 (APC‐Cy7), anti‐CD38‐APC and 7AAD (all from BD Biosciences). Fluorescence intensity was measured on a BD FACSCanto II flow cytometer and analysed using FlowJo (version 10) software.
5
PBMC Isolation and Functional Assay
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PBMC were isolated from fresh blood using Ficoll and counted using Luna-FL cell counter (Logos Biosystems). Frozen PBMCs were thawed, counted and 1 × 106 PBMCs were stimulated with MVA-Venus (MOI = 1) for 24 h in a total volume of 200 µl of RPMI media with 5% of human serum in a 96 wells plate. Unstimulated cells and PHA (20 µg/ml) were used as negative and positive controls, respectively. Anti-CD107a-APC, Golgi Plug (Brefeldin A) and Golgi Stop (Monesin) were added 4 h before staining. Cells were washed with PBS and stained 15 min at room temperature with Live and dead near IR, anti-CD3-FITC (Biolegend 300440, 1:40), anti-CD4-BV421 (BD 566703, 1:50), anti-CD8-BV605 (Biolegend 301040, 1:100), anti-CD69-PE (BD 555531, 1:10), anti-CCR7-PE/Dazzle 594 (Biolegend 353236, 1:20) and anti-CD45RA-PerCP (Biolegend 304062, 1:20). After washing, cells were permeabilized with Fix/Perm (BD) for 20 min at 4 °C. Cells were washed with Perm/Wash (BD) and intracellular stained with anti-IFNγ-PECy7 for 30 minutes at 4 °C, washed and resuspended in FACS buffer until acquisition. Cells were acquired in Attune NxT cytometer (Thermofisher) and data was analyzed with Flow Jo X software following the gating strategy described in Supplementary Fig. 2 . Only two out of five DL3 volunteers had available PBMC samples for the analysis.
6
Phenotypic Analysis of T-cells by Flow Cytometry
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Flow cytometry analysis of T-cell phenotype was performed according to the protocols as previously described. [21] [22] [23] Monoclonal antibodies used in this study including anti-CD3-BV605, anti-CD45-PE, anti-CD4-BV421, anti-CD8-APC-R700 anti-CD95-DX2, anti-CD28-CD28.2, anti-HLA-DR-PE-Cy7, anti-CD69-APC and anti-CCR5-PE, were purchased from BD Biosciences (San Jose, CA, USA). Anti-CD38-FITC was obtained from StemCell Technologies (Vancouver, BC, Canada). Anti-CD25-APC was from Biolegend (San Diego, CA, USA), and anti-CD127-PE was a product of Invitrogen (Carlsbad, CA, USA). CD4 + T cell differentiation was identi ed in terms of CD28 and CD95 expression, as CD28 + CD95 + CD4 + T cell de ned as central memory CD4 + T cell (CD4 + Tcm) and CD28 + CD95 -CD4 + T cell as effector memory CD4+ T cell (CD4 + T EM ). CD28 + CD95 + CD8 + T cell was de ned as CD8 + T CM cell whereas CD28 + CD95 -CD8 + T cell was CD8 + T EM cell. In addition, expression of CD25 and CD127 were measured to evaluate regulatory T Cells (Tregs). Activation markers HLA-DR, CD38 and CD69 were measured on CD4 + and CD8 + T cells, and CD4 + CCR5 + T cells were de ned as SIV-infected cells. All dates were acquired and analyzed on a ow cytometer (FACSCanto; Becton Dickinson).
7
Phenotypic Analysis of CAR T Cell Therapy
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At experimental endpoint of the intracranial tumor experiments in which mice were treated with CAR T cells, some organs were taken for analysis by flow cytometry. The brain, spleen, lymph nodes and blood (in the form of a cardiac bleed) were isolated from mice from each treatment group (n = 3 or 4 mice per group). Organs were processed through a 70‐µm filter to a single‐cell suspension and prepared for antibody staining. Blood was collected in a heparin/EDTA tube. Red blood cells were lysed by incubation in red cell removal buffer (156 mm ammonium chloride, 11.9 mm sodium bicarbonate, 0.097 mm EDTA, WEHI, Melbourne) and centrifugation at 548RCF for 5 min at 4°C. Cells were labelled with a cell surface murine antibody cocktail containing anti‐CD3‐AF700 (BD Pharmingen, New Jersey), anti‐CD4‐BV421 (BD Horizon, New Jersey), anti‐CD8‐BV711 (BioLegend, San Diego), anti‐PD‐1 (BD Pharmingen, New Jersey), LAG‐3 (CD223)‐APC (BD Pharmingen, New Jersey), anti‐CD27‐BV605 (BioLegend, San Diego), anti‐CD28‐PE‐Cy7 (eBioscience, San Diego), CD44‐BV786 (BD Horizon, New Jersey) and anti‐CD62L‐BV510 (BD Horizon, New Jersey). Samples were analysed using a BD Fortessa X20 flow cytometer and FlowJo™ software v10 (BD, New Jersey).
8
Cytokine profiling of CAR T cells
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CAR T cells and tumour cells were co-incubated for 5 h in T cell media at a 1:1 ratio in the presence of Golgi-stop (Becton Dickinson, Kit #554715, final concentration 5 ml/mL), at 37 °C, 5% CO2. Cells were subsequently incubated with antibodies to surface proteins in FACS buffer (Phosphate Buffered Saline (PBS) with 0.2% Bovine Serum Albumin (BSA, Gibco)), with anti-CD4-BV421 (Clone MP6-XT22, BD Horizon, Franklin Lakes, NJ, USA), anti-CD8-BV711 (Clone 53-6.7, BioLegend, San Diego, CA, USA), and the viability dye Fixable yellow (Invitrogen, Oregon) for 30 min at 4 °C. Cells were then fixed for 20 min at room temperature and permeabilised using BD Pharmingen Fix/Perm kit (Cat: 554715, BD Pharmigen, Franklin Lakes, NJ, USA) according to manufacturer’s instructions. Intracellular IFNγ, IL-2 and TNFα were detected by incubating with anti-IFNγ-APC (Clone XMG1.2, BD Pharmingen, Franklin Lakes, NJ, USA), anti-IL2-PE (Clone JES6-5H4; BD Biosciences, East Rutherford, NJ, USA), and anti-TNFα-AF488 (Clone MP6-XT22; Biolegend) antibodies for 1 h at 4 °C. Cells were washed with a FACS buffer and analysed using a Fortessa X20 (BD Biosciences, East Rutherford, NJ, USA) and analysis performed on FlowJo software V10.8 (TreeStar, Ashland, OR, USA).
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