Treg were stimulated with rhIL-2 (25 U/mL) alone as control, or together with either soluble CD28 agonist mAb (1 μg/mL, Clone L293, Cat# 348040; BD Bioscience), plate-bound CD3 mAb (5 μg/mL, Clone UCHT1, BD Bioscience), plate-bound CD3 (5 μg/mL) plus soluble CD28 mAb (1μg/mL), CD28-superagonist ANC28.1 (1 μg/mL, Clone ANC28.1/5D10, Cat# 177–820, preservative free; Ancell, Bayport, USA), or anti-CD3/anti-CD28 mAb-coated microbeads (Invitrogen by Life technologies, Bleiswijk, The Netherlands) used in a 1:2 bead-to-cell ratio. Titration of soluble CD28-agonist and CD28-superagonist antibodies at a concentration range of 1–10 μg/mL led to similar FOXP3 expression levels as determined by flow cytometry. Cells were cultured in 96-well round bottom plates in RPMI 1640 (Invitrogen) as described previously44 (link). In selected experiments immunomodulating agents were analyzed; Treg were pretreated with wortmannin (5 μM), rapamycin (200 nM) (Sigma-Aldrich, St. Louis, USA)15 (link), or vehicle control for 30 min at 37 °C. Thereafter, stimulators as indicated were added to the culture mixture.
Clone l293
Manufactured by BD
Sourced in United States
The Clone L293 is a dual H-bridge motor driver integrated circuit capable of driving two DC motors or one stepper motor. It is designed to provide bidirectional control of small DC motors.
Automatically generated - may contain errors
Lab products found in correlation
Rapamycin, by Merck Group (1 mentions)
Wortmannin, by Merck Group (1 mentions)
Clone ucht1, by BD (1 mentions)
Anti cd3 anti cd28 mab coated microbeads, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
96 well round bottom plate, by Corning (1 mentions)
Brefeldin a, by BD (1 mentions)
Monensin, by BD (1 mentions)
3 protocols using clone l293
1
Regulatory T Cell Activation Assay
2
PBMC Stimulation with ESAT-6 and PPD
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PBMCs were assessed for ex vivo responses to peptide pools of Early Secretory Antigen Target-6 (ESAT-6) (2 µg/mL) and Purified Protein Derivate (PPD) (10 µg/mL), both provided by Statens Serum Institute, Copenhagen, Denmark. Staphococcal Enterotoxin B (SEB; Sigma) and PBS were used for positive and negative controls, respectively. Approximately 1 million PBMCs in a total volume of 300 µL were stimulated for 6 hours in 96-well round bottom plates (Corning Life Sciences) in the presence of αCD28 (10 µg/mL, clone L293, BD) and αCD49d (10 µg/mL, clone L25, BD). Samples from the same subject were set up side by side in the same plate. Brefeldin A (1.0 µl/well) and monensin (0.7 µl/well) (both from BD) were added to the cell-antigen mix prior to the 6 h incubation.
3
T-cell Proliferation Assay with LDN
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T cells were labeled with 1 µM Carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes, Life Technologies; Cat.no: V12883) at 37°C for 10 min, followed by quenching in 10% FBS-RPMI 1640 (Lonza, Cat.no: 12-167F) for an additional 10 min. T cells (1 × 105) were plated in 96-well round-bottom plates bound with affinity-purified antibody goat anti-mouse IgG(H+L) Human Serum Adsorbed (10ug/mL; SeraCare's KPL, Mildford, MA; Cat.no: 5210-0185), soluble anti-CD3 (0·5 µg/mL; Clone OKT3 (Functional Grade); eBioscience; Cat.no: 16-0037-85) and soluble anti-CD28 antibodies (0·25 µg/mL; Clone L293; BD Biosciences; Cat.no: 348040). Autologous LDN (1 × 105) were added in a final volume of 200 uL for a co-culture ratio of 1:1 (T-cells:LDN). Positive and negative controls included T cells alone with or without anti-CD3 and anti-CD28 antibodies, respectively. All cultures were incubated for 72 h at 37°C, 5% CO2 after which proliferation was tested by CFSE fluorescence dilution using a Gallios flow cytometer. Data are expressed as a percent proliferation relative to positive T cells control, calculated as follows: number of proliferating T-cells in co-culture T-cells:LDN x 100 / number of proliferating T cells in positive control represented as T-cells:LDN ratio 1:0.
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