An untreated, flat-bottom, 96-well plate was coated overnight at 4°C with 0.2 μg/mL anti-CD3 (clone 145-2C11, BD Biosciences) and 0.5 μg/mL anti-CD28 (clone 37.51, BD Biosciences) in PBS, washed with PBS, and blocked with coculture media (RPMI from Gibco containing 10% FBS from Atlanta Biologicals, 1% penicillin/streptomycin from Gibco, and 1× β-mercaptoethanol from Gibco) for at least 30 minutes at room temperature (RT). CD8+ T cells were isolated from the spleens of naive C57BL/6 mice using the untouched CD8+ T cell isolation kit (Miltenyi Biotec), following manufacturer’s instructions. Isolated CD8+ T cells were washed twice with PBS and stained with CTV dye (Life Technologies) following manufacturer’s instructions. Dye-labeled CD8+ T cells were then cultured on the anti-CD3/anti-CD28–coated plate at a density of 105 cells per well in coculture media, and 2 μg/mL anti–IL-2 (using a mixture of both S4B6-1 and JES6-1A12 clones, Bio X Cell) was added at the beginning of coculture where indicated. The cells were cultured at 37°C and 5% CO2 for 3 days. Following coculture, cells were stained and analyzed by flow cytometry.
Anti cd28 clone 37
Manufactured by BD
Sourced in United States
Anti-CD28 (clone 37.51) is a monoclonal antibody that binds to the CD28 surface receptor expressed on T cells. Its core function is to provide co-stimulatory signals that can enhance T cell activation and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3 clone 145 2c11, by BD (14 mentions)
Anti cd3ε clone 145 2c11, by BD (4 mentions)
Golgiplug, by BD (4 mentions)
Cytofix cytoperm kit, by BD (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Ctv dye, by Thermo Fisher Scientific (2 mentions)
Untouched cd8 t cell isolation kit, by Miltenyi Biotec (2 mentions)
Anti il 2, by BioXCell (2 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (2 mentions)
Ifnγ clone xmg1, by BioLegend (2 mentions)
Cd4 clone rm4 5, by BioLegend (2 mentions)
Cd8 clone 53 6, by BioLegend (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Anti cd3 clone hit3α, by BD (2 mentions)
Easysep human cd3 and cd8 t cell isolation kit, by STEMCELL (2 mentions)
Easysep mouse cd3 and cd8 t cell isolation kit, by STEMCELL (2 mentions)
33 protocols using anti cd28 clone 37
1
Expansion of CD8+ T Cells In Vitro
2
Multiparameter Flow Cytometry Analysis
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Fluorescein isothiocyanate (FITC), Allophycocyanin cyanine tandem (APC-H7), R-phycoerythrin (PE) or Allophycocyanin (APC)-conjugated monoclonal antibodies (mAbs) were used for cytofluorometric analysis of anti-mouse Ki67 (BD PharMingen, San Diego, CA, USA), anti-mouse CD4, anti-mouse CD25 and anti-mouse FoxP3 (eBioscience, San Diego, CA). Purified hamster anti-mouse mAbs, anti-CD3 (clone 145-2C11) and anti-CD28 (clone 37.51) were also purchased from BD Pharmingen. Recombinant TGF-β was purchased from Peprotech (NJ, USA). TGF-β neutralizing mAb (1D11) was a gift from Dr. Hong-Ming Hu (Earle A. Chiles Research Institute, Portland, OR). Cell enrichment kits for CD4+ and Antigen Presenting Cells (APC, CD90.1−) were purchased from MACS Miltenyi Biotec Inc., (Auburn, CA, USA). Dead Fixable Violet Dead Cell Stain Kit was purchased from Invitrogen (L34955, Carlsbad, CA).
3
MDSC-Mediated T Cell Suppression
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Murine T cells were enriched from wild-type mouse splenocytes by negative selection using a murine pan-T-cell isolation kit (EasySep, StemCell). To monitor T-cell proliferation, isolated T cells were stained with 2.5 µM CellTrace Violet (Invitrogen) according to the manufacturer’s instructions.
Murine MDSCs were isolated from the splenocytes of tumor-bearing mice by negative selection using a murine MDSC isolation kit (EasySep, StemCell). As indicated, the obtained MDSCs were used immediately or pretreated with sialidase as described below.
Isolated MDSCs and T cells were plated at a ratio of 1:1 in a 96-well flat bottom plate and cocultured for 48 h in complete RPMI in the presence of 50 IU of IL-2 (proleukin). For T-cell stimulation, the plate was coated with anti-CD3 (clone 17A2; BioLegend) and anti-CD28 (clone 37.51; BD Biosciences) antibodies. The supernatants were frozen at −80 °C, and the cells were stained for flow cytometry.
For Siglec-E blocking, purified anti-mouse Siglec-E antibody (M1305A02; BioLegend) or rat IgG2a or κ isotype control (BioLegend) was added at a final concentration of 10 µg/mL. CCL2 blocking was performed by the addition of 50 µg/mL CCL2 (Clone 2H5, BD).
Murine MDSCs were isolated from the splenocytes of tumor-bearing mice by negative selection using a murine MDSC isolation kit (EasySep, StemCell). As indicated, the obtained MDSCs were used immediately or pretreated with sialidase as described below.
Isolated MDSCs and T cells were plated at a ratio of 1:1 in a 96-well flat bottom plate and cocultured for 48 h in complete RPMI in the presence of 50 IU of IL-2 (proleukin). For T-cell stimulation, the plate was coated with anti-CD3 (clone 17A2; BioLegend) and anti-CD28 (clone 37.51; BD Biosciences) antibodies. The supernatants were frozen at −80 °C, and the cells were stained for flow cytometry.
For Siglec-E blocking, purified anti-mouse Siglec-E antibody (M1305A02; BioLegend) or rat IgG2a or κ isotype control (BioLegend) was added at a final concentration of 10 µg/mL. CCL2 blocking was performed by the addition of 50 µg/mL CCL2 (Clone 2H5, BD).
4
Proliferation Assay for Splenocyte Activation
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Proliferation assays were performed as previously described
[15 (link)]. In brief, splenocytes were isolated from C57BL/6 adult mice or fetal sheep and seeded in 96-well, flat-bottom microtiter plates (Nunc, Thermo Fisher Scientific, Australia). Assays were performed in triplicate at a concentration of 2.5 × 105 cells per well in complete RPMI medium alone or in the presence of 5 μl/ml concanavalin A (ConA; Sigma-Aldrich), or into wells precoated with 10 μg/ml anti-CD3 (clone 145-2C11) and 10 μg/ml anti-CD28 (clone 37.51) antibodies (both from BD Biosciences) to a final volume of 200 μl per well. For wells that required addition of hAECs, 50 μl of hAECs that had been exposed to surfactant or PBS, at hAEC-to-splenocyte ratios ranging from 1:5 to 1:40, were added to each well before the addition of splenocytes. Cells were incubated at 37°C for 48 hours and then 1 μCi/well [3H]-thymidine (Perkin Elmer, Waltham, MA, USA) was added for an additional 18 hours of culture. Cells were harvested onto filter mats (Perkin Elmer), and incorporated radioactive nucleic acids were counted by using a Top Count Harvester (Packard Biosciences, Meriden, CT, USA).
[15 (link)]. In brief, splenocytes were isolated from C57BL/6 adult mice or fetal sheep and seeded in 96-well, flat-bottom microtiter plates (Nunc, Thermo Fisher Scientific, Australia). Assays were performed in triplicate at a concentration of 2.5 × 105 cells per well in complete RPMI medium alone or in the presence of 5 μl/ml concanavalin A (ConA; Sigma-Aldrich), or into wells precoated with 10 μg/ml anti-CD3 (clone 145-2C11) and 10 μg/ml anti-CD28 (clone 37.51) antibodies (both from BD Biosciences) to a final volume of 200 μl per well. For wells that required addition of hAECs, 50 μl of hAECs that had been exposed to surfactant or PBS, at hAEC-to-splenocyte ratios ranging from 1:5 to 1:40, were added to each well before the addition of splenocytes. Cells were incubated at 37°C for 48 hours and then 1 μCi/well [3H]-thymidine (Perkin Elmer, Waltham, MA, USA) was added for an additional 18 hours of culture. Cells were harvested onto filter mats (Perkin Elmer), and incorporated radioactive nucleic acids were counted by using a Top Count Harvester (Packard Biosciences, Meriden, CT, USA).
5
Splenocyte Activation and Expansion Protocol
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Untreated, flat-bottom, 24-well plates were coated overnight at 4°C with 0.2 μg/mL anti-CD3 (clone 145-2C11, BD Biosciences) and 0.5 μg/mL anti-CD28 (clone 37.51, BD Biosciences) in PBS, washed with PBS, and blocked with coculture media for at least 30 minutes at RT. Erythrocytes were removed with ACK lysing buffer (Gibco), and splenocytes were plated at 1.2 × 106 per well. Splenocytes were activated for 96 hours before being moved to 6-well plates, and fresh medium was added at a 1:1 ratio. Splenocytes were allowed to expand further 72 hours.
6
Profiling Tregs and Tconv by Proteomics
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Tregs and Tconv were profiled by proteome and quantitative phosphopeptide sequencing (van Ham et al., under preparation). In brief, CD4+CD25+ Tregs and CD4+CD25– Tconv were ex vivo isolated from single cell suspensions of pooled spleen and lymph node (LN) cells from BALB/c mice by MACS-based enrichment of CD4+ T cells using direct beads (L3T4, Miltenyi Biotec) followed by flow cytometry-based sorting to high purity. For proteome analysis, sorted T cell subsets were left unstimulated. For quantitative phosphopeptide sequencing, cells were either left unstimulated or stimulated by decoration with biotinylated anti-CD3 (clone145-2C11, BD Biosciences) and anti-CD28 (clone 37.51, BD Biosciences) and subsequent antibody crosslinking using streptavidin. Stimulation was stopped after 5 min with an excess of ice cold PBS and cells were further processed for liquid chromatography – tandem mass spectrometry (LC–MS/MS) (further experimental details available on request).
7
Isolation and Activation of CD4+ T and B Cells
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Naive CD4+ T and B cells were purified by negative selection using MACS systems (Miltenyi Biotec). CD4+PD1+CXCR5+ cells were sorted by FACS after staining with FITC-conjugated PD1, phycoerythrin-conjugated CD4, and PerCP-conjugated CXCR5 antibodies. Purified CD4+ T cells were stimulated with plate-bound anti-CD3 (clone 145-2C11, BD Biosciences) at 5 μg/ml or at the indicated concentrations and anti-CD28 (clone 37.51, BD Bioscience) (2 μg/ml) antibodies for the indicated times. B cells were activated with soluble anti-F(ab′)2 fragments of goat anti-mouse IgM (anti-IgM F(ab′)2, Jackson ImmunoResearch Laboratories, Inc.) at 5 μg/ml for the indicated times.
8
Cultivation and Differentiation of Melanoma Cell Lines and CD4+ T Cells
9
Analyzing T-cell Cytokine Production
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Lung cells were incubated for 1 h in the presence of 1 μg/ml anti-CD28 (clone 37.51; BD Pharmingen) and anti-CD49d (clone 9C10 (MFR4.B); BD Pharmingen) followed by 5 h with 10 μg/ml brefeldin A (Sigma-Aldrich) and 1:1500 BD GolgiStop (BD Biosciences) at 37°C in an automated heater that cooled the cells to 4°C after incubation. The next day, cells were washed in FACS buffer (PBS containing 0.1% sodium azide and 1% FCS) and stained at 4°C for surface markers using anti-CD4-APC-eF780 (clone RM4-5; 1/600 dilution; eBioscience) and anti-CD44-FITC (clone IM7; 1/200 dilution; BD Bioscience). After 15–30 min of surface staining, cells were washed in FACS buffer, permeabilized using the Cytofix/Cytoperm kit (BD Pharmingen) according to the manufacturer’s instructions, and stained intracellularly with anti-IL-17-PerCP-Cy5.5 (clone eBio17B7; 1/200 dilution; eBioscience) and anti-IFNγ-PE-Cy7 (clone XMG1.2; 1/200, eBioscience) mAbs. Cells were subsequently washed, resuspended in FACS buffer and analyzed on a BD FACSCanto flow cytometer (BD Biosciences). In vivo staining with anti-CD45.2: Anti-CD45.2-FITC (clone 104; BD Pharmingen) was diluted to 10μg/ml in sterile Phosphate-buffered saline (PBS). 250μl of the aCD45.2 solution was injected i.v. into each mouse via the tail vein, three minutes before euthanization of the mice. Data was analyzed using FlowJo v10.0.7 for Windows.
10
IFN-γ ELISpot Assays for Influenza A
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IFN-γ ELISpot assays were performed using a commercial kit from Abcam (ab64029) following the manufacturer’s recommendations. Cells were stimulated with 1.25 μg/ml anti-CD28 (Clone 37.51, BD) and premade 15-mer sequences with 11 amino acids overlap peptides from influenza A (H1N1) HA (Peptivator, Miltenyi Biotech) or NA (PepMixTM, JPT). Spots were counted using the AID iSpot reader and ELISpot Reader software V 7.0.
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