Total spleen cells from either Foxp3EGFPCre or Foxp3EGFPCreR26N1c/N1c mice were stimulated at 37°C in nonsupplemented RPMI 1640 using preformed complexes of biotinylated anti-CD3 mAb (clone 145-2C11, BD, 30 μg/mL), anti-CD4 mAb (GK1.5, BD, 30 μg/mL), and streptavidin (60 μg/mL) for 1–5 minutes. The reaction was stopped, and cells were permeabilized using Foxp3 transcription factor staining buffer (eBiosciences) and Perm Buffer III (BD Biosciences). Cells were stained with the antibodies described in Supplemental Table 2 . The samples were acquired on a BD Fortessa cytometer, and data were analyzed with FlowJo software.
Anti cd3 mab clone 145 2c11
Manufactured by BD
Sourced in United States
Anti-CD3 mAb (clone 145-2C11) is a mouse monoclonal antibody that specifically binds to the CD3 complex on the surface of T cells. It is used as a research tool in immunology studies.
Automatically generated - may contain errors
Lab products found in correlation
Foxp3 transcription factor staining buffer, by Thermo Fisher Scientific (2 mentions)
Perm buffer 3, by BD (2 mentions)
Facsvantage, by BD (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Soluble anti cd28 mab clone 37.51, by BD (1 mentions)
3h tdr, by PerkinElmer (1 mentions)
Microbeta filtermate 96 harvester, by PerkinElmer (1 mentions)
Microbeta2 plate counter, by PerkinElmer (1 mentions)
P p65 s536, by Thermo Fisher Scientific (1 mentions)
Fortessa cytometer, by BD (1 mentions)
Celltrace cfse, by Thermo Fisher Scientific (1 mentions)
Anti cd3 mab, by BD (1 mentions)
Scintillation counter, by PerkinElmer (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Clariostar microplate reader, by BMG Labtech (1 mentions)
Mars data analysis software, by BMG Labtech (1 mentions)
Protein 200 array reader, by Bio-Rad (1 mentions)
Murine multiplex cytokine kit, by Bio-Rad (1 mentions)
Manager software 4, by Bio-Rad (1 mentions)
Golgistop solution, by BD (1 mentions)
10 protocols using anti cd3 mab clone 145 2c11
1
Foxp3 Regulatory T Cell Profiling
2
Murine Lymphocyte Proliferation Assay
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Murine spleen and skin lymphocytes with or without CD25 depletion were loaded with the fluorescent cytoplasmic dye carboxyfluorescein diacetate, succinimidyl ester (CFSE), as described (Invitrogen), and stimulated with plate bound anti-CD3 mAb (clone 145-2C11, BD Pharmingen). After 3 days, cells were stained with anti-CD4-APC mAb (BD) and analyzed on a FACSVantage. Percent of input cells that proliferated were calculated, as described [19] .
3
T Cell Proliferation Assay with 1,25(OH)2D3
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Purified T cells were plated into a 96-well round-bottom cell culture plate (2 × 105 cells/well; Thermo Fisher Scientific, Waltham, MA, USA), cultured with or without 5 μg/mL plate-bound anti-CD3 mAb (clone 145-2C11; BD Biosciences) and 2 μg/mL soluble anti-CD28 mAb (clone 37.51; BD Biosciences), and treated with 10 nM 1,25(OH)2D3 or a vehicle control for 72 h. Cells were pulsed with 0.5 uCi of [3H] TdR (PerkinElmer, Waltham, MA, USA) in 20 μL of complete RPMI for the last 8 h. The cells were harvested on filter paper using a MicroBeta FilterMate-96 Harvester (PerkinElmer), and the proliferation was quantified by measuring the amount of [3H] TdR incorporated into the DNA, as determined by the MicroBeta2 Plate Counter (PerkinElmer). Data are expressed as counts per minute.
4
Foxp3 and Stk3/4 Phosphorylation Assay
5
T Cell Proliferation Assay with CFSE Labeling
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WT and lamp2 KO-derived CD4 and CD8 T cells were sorted, labeled with 1 μM of CellTraceTM CFSE (carboxyfluorescein diacetate succinimidyl ester; ThermoFisher Scientific, C34554) and cultured in the presence of 1 μg/ml anti-CD3 mAb (clone 145–2C11; BD Pharmingen, 553,057) and 5 μg/ml anti-C28 mAb (clone 37.51; BD Pharmingen, 553,294). The precursor frequency of dividing cells (percentage of cells in the initial population that undergone one or more divisions after culture) was calculated as follows: [∑n ≥ 1(Pn/2 n)]/[∑n ≥ 0(Pn/2 n)], where n is the division number that cells have gone through and Pn is the number of cells in division, as described [56 (link)].
6
Splenocyte Cytokine Production and Proliferation
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The ability of the splenocyte cultures to produce cytokines in response to a mitogenic challenge was assessed by incubating total splenocyte cultures (5 × 106 cells/mL) for 48 h (at 37°C, 5% CO2, and 90% humidity) in the presence of 0.25 μg/mL concanavalin A (con A, Perkin Elmer Life Science, Boston, MA, USA) or 0.1 μg/mL anti-CD3 mAb (clone 145-2C11, BD Pharmingen, San Diego). After that period, the supernatants were harvested, aliquoted, and stored at −20°C until being assayed for cytokines. A second portion of the splenocyte suspension was placed in a 96-well U-bottomed plate (Becton Dickinson, Franklin Lakers, NJ, USA) in aliquots of 100 μL. The cells' ability to proliferate in response to mitogenic stimulation with 0 (negative control), 0.25 μg/mL con A, or 0.1 μg/mL anti-CD3 mAb was determined by incubation for 48 h at 37°C in a 5% CO2 atmosphere with 90% humidity. The cultures were pulsed with [3H] thymidine (0.5 μCi/well) for the last 18 to 24 h of incubation, cells were harvested, and radioactivity of [3H] thymidine incorporation was counted using a scintillation counter (Perkin Elmer Life Science, Boston, MA). [3H] Thymidine uptake was expressed as the mean counts per minute (c.p.m.) of sextuplicate wells.
7
T Cell Activation Assay Protocol
8
Cytokine Profiling of Activated PD-1+ T Cells
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Flow sorted PD-1+ or PD-1− T cells from 5T33 myeloma-bearing mice were activated with plate-bound anti-CD3 mAb (clone 145-2C11, BD Biosciences; 5 μg/mL). Culture supernatants were harvested after 48 h and stored at −80 °C. Thawed supernatants were then analyzed using a murine multiplex cytokine kit (Bio-Rad, Hercules, CA) to detect IL-2, IL-4, IL-5, IL-10, IL-12p70, granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-α), and IFN-γ. Cytokines were quantified using a Bio-Plex protein 200 array reader, and data was automatically processed and analyzed using Bio-Plex Manager Software 4.1. Standard curves were generated from recombinant cytokine standards. All samples were assayed in duplicate.
9
Quantifying Murine Splenic Cytokines
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On Experimental Day 6, spleen cells of each group of mice were weighed with an electronic balance and were then extracted via mechanical disruption using 30 μm steel mesh screens in a fresh Petri dish to prepare single-cell suspensions. For intracellular detection of cytokines, 2 × 105 cells/well were stimulated with plate-bound anti-CD3 mAb [clone 145-2C11 (BD Pharmingen, Oxford, UK); 5 μg/mL] and soluble anti-CD28 (1 μg/mL) for 24 hours. Golgistop solution (BD Biosciences, San Diego, CA, USA) was put into the culture 4 hours before cell harvesting. The cells were then stained with phycoerythrin-conjugated antimouse CD4 (GK1.5; BioLegend), followed by treatment with Cytofix/Cytoperm Plus Kit (BD Biosciences) according to the manufacturer’s instructions (BD Biosciences). Cells were then stained intracellularly using the fluorescein isothiocyanate (FITC)-conjugated mAbs specific to murine IL-17A (TC11-18H10.1) and IL-22 (Poly5164), purchased from BioLegend. The stained cells were acquired on a BD Accuri C5 cytometer Cat. No. 657214 (BD Biosciences, San Jose, CA, USA) and analyzed with BD Accuri C6 software, version 1.0264.21.
10
Sorting and Expansion of PD-1+ T Cells from Myeloma Mice
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Sorting of PD-1+ or PD-1− T cells from 5T33-myeloma bearing mice was performed using a FACSAria flow cytometric sorter. T cells were activated and expanded in culture with plate-bound anti-CD3 mAb (clone 145-2C11, BD Biosciences; 5 μg/mL) and anti-CD28 mAb (clone 37.51, BD Biosciences; 1 μg/mL) in the presence of IL-2 (20 U/ml), IL-7 (5 ng/ml) and IL-15 (5 ng/ml) for 7 days.
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