PBMCs were isolated within 12 h of sample collection and monocytes were removed by overnight plating in a culture dish. The next day, the CD4+CD45RA+ cells were purified using naïve CD4+ T-cell isolation kit II (product no. 130-094-131, Miltenyi Biotec). CD4+ T-cells were isolated using positive selection magnetic beads using isolation kit (product no. 130-045-101, Miltenyi Biotec). Purified naïve CD4+ T-cells represented a >97% pure population. Purified cells were maintained in culture with 20 U of IL-2 for two days. Thereafter, these cells were stimulated with plate-bound ICs at 10 μg/ml and purified soluble C5b-9 at 2.5 μg/ml for each 1X106 cells in the presence of plate-bound anti-CD3 at 0.25 μg/ml. Positive control cells were stimulated with plate-bound 2 μg/ml of anti-CD28 (clone 28.2) and 0.25μg/ml of anti-CD3 (eBioscience, clone OKT3). For inhibition, cells were cultured in 25nM of P505, a Syk inhibitor (product no. PRT06207, Sellkchem) and 50 μM of HCQ (product no. 263010250, Acros organics). Cells were cultured for 48 h in the presence of IL-2 (20 IU), for each one ml of medium (Peprotech). Post 48 h, cells were re-stimulated and processed for staining at 96 h.
Anti cd28 clone 28
Manufactured by Thermo Fisher Scientific
The Anti-CD28 (clone 28.2) is a monoclonal antibody that recognizes the CD28 receptor on the surface of T cells. CD28 is a co-stimulatory molecule that plays a crucial role in the activation and proliferation of T cells. The Anti-CD28 (clone 28.2) can be used in various in vitro applications to study T cell biology and function.
Automatically generated - may contain errors
Lab products found in correlation
Easysep human cd4 t cell enrichment kit, by STEMCELL (3 mentions)
Dynabeads regulatory cd4 cd25 t cell kit, by STEMCELL (3 mentions)
Anti cd3 okt3, by Thermo Fisher Scientific (3 mentions)
Clone okt3, by Thermo Fisher Scientific (1 mentions)
Na ve cd4 t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Foxp3 staining buffer, by Thermo Fisher Scientific (1 mentions)
Anti cd3 clone okt3, by Thermo Fisher Scientific (1 mentions)
Facs caliber, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Roswell park memorial institute (rpmi), by Lonza (1 mentions)
Anti foxp3 clone 206d, by BioLegend (1 mentions)
96 well round bottom plate, by Corning (1 mentions)
Anti cd3 ucht1, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Pe cy7, by BD (1 mentions)
Whole mouse igg, by Jackson ImmunoResearch (1 mentions)
Anti cd3 antibody clone okt3, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Corning (1 mentions)
7 protocols using anti cd28 clone 28
1
Naïve CD4+ T-cell Stimulation Assay
2
Isolation and Expansion of Human Tregs
3
Expansion and Purification of Human Regulatory T Cells
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Freshly isolated, blood-derived T cell populations were cultured in 96-well round bottom plates (Costar) coated with 0.5 ug/ml anti-CD3 (UCHT-1, eBioscience) with irradiated antigen presenting cells (iAPC, PBMCs depleted of T cells via anti-CD2-beads from Dynal/Invitrogen) and 0.25 ug/ml anti-CD28 (clone 28.2 eBioscience) in RPMI-1640 medium (Life Technologies) supplemented with 10% FBS (Life Technologies) and 20 U/ml rhIL-2. Confluent wells were split into new plates and IL-2 containing media was supplemented every 2–3 days. After two weeks, purity (expression of FoxP3high) was tested by flow cytometry using anti-FoxP3 (clone 206D, BioLegend) and the intracellular FoxP3 staining kit (Biolegend).
4
In vitro Generation of Regulatory T Cells
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Example 7
Treg cells were generated in vitro using the following method. CD4+ enriched T cells (isolated using the EasySep™ Human CD4+ T Cell Enrichment Kit from Stemcell Technologies) from PBMC were incubated with anti-CD3/anti-CD28 beads (20 μl beads in 100 μl volume, or 4:1 beads to cell ratio using Dynabeads® Regulatory CD4+CD25+T Cell Kit) with 500 U/mL of IL2 in the presence of 0.1 μg/mL rapamycin for a week. Cells were replaced with new culture with anti-CD3 (OKT3, eBiosciences) plate bound at 0.5 μg/mL and soluble 0.5 μg/mL anti-CD28 (clone 28.2, eBiosciences) with 100 U/mL of IL2 and 0.1 μg/mL rapamycin.
Induction of Treg cells was assayed using the alamar blue cell viability assay in the presence of anti-CD4×IL2 Fc-fusions or control antibodies. Results are shown in
5
In Vitro Generation of Regulatory T Cells
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Example 7
Treg cells were generated in vitro using the following method. CD4+ enriched T cells (isolated using the EasySep™ Human CD4+ T Cell Enrichment Kit from Stemcell Technologies) from PBMC were incubated with anti-CD3/anti-CD28 beads (20 μl beads in 100 μl volume, or 4:1 beads to cell ratio using Dynabeads® Regulatory CD4+CD25+ T Cell Kit) with 500 U/mL of IL2 in the presence of 0.1 μg/mL rapamycin for a week. Cells were replaced with new culture with anti-CD3 (OKT3, eBiosciences) plate bound at 0.5 μg/mL and soluble 0.5 μg/mL anti-CD28 (clone 28.2, eBiosciences) with 100 U/mL of IL2 and 0.1 μg/mL rapamycin.
Induction of Treg cells was assayed using the alamar blue cell viability assay in the presence of anti-CD4×IL2 Fc-fusions or control antibodies. Results are shown in
6
T Cell Differential Stimulation Assay
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Purified T cells (1 × 106) were differentially stimulated using anti-CD3 antibody (clone OKT3; eBioscience; 16-0037085; 0.1 μg/mL), anti-CD28 (clone 28.2; eBioscience; 16-0289-85; 2.5 μg/mL), and/or CD81 (clone 5A6; generated in the laboratory of S.L.; 2.5 μg/mL). Antibodies were then cross-linked using goat F(ab)2 anti-mouse immunoglobulin G (IgG) (Jackson ImmunoResearch; 115-006-062; 4 μg/mL). Unstimulated T cells were used as a control. T cells were cultured in RPMI1640 with l -glutamine (Corning Cellgro; 10-040-CV) supplemented with 10% heat-inactivated donor calf serum (HyClone; GE Healthcare Life Sciences; SH30109.03), l -glutamine (Mediatech; 25-005-Cl), and penicillin:streptomycin (Gemini Bio-Products; 400-109) and incubated for 24 and 48 h. Following incubation, cells were washed with phosphate-buffered saline containing 1% bovine serum albumin and blocked by 50 μg of whole-mouse IgG (Jackson ImmunoResearch; 015-000-003) for 20 min on ice (to prevent unbound goat anti-mouse cross-linkers from nonspecifically binding to mouse anti-human phenotyping antibodies). Following blocking, cells were washed and then stained for surface expression of CD4 (Pacific Blue; BD Pharmingen; 558116), CD8 (fluorescein isothiocyanate; BD; 347313), CD62L (APC; BD Pharmingen; 559772), CD45RO (PE-Cy7; BD; 337168), and CD69 (PE; BD; 341652).
7
In Vitro Generation of Regulatory T Cells
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Example 7
Treg cells were generated in vitro using the following method. CD4+ enriched T cells (isolated using the EasySep™ Human CD4+ T Cell Enrichment Kit from Stemcell Technologies) from PBMC were incubated with anti-CD3/anti-CD28 beads (20 μl beads in 100 μl volume, or 4:1 beads to cell ratio using Dynabeads® Regulatory CD4+CD25+T Cell Kit) with 500 U/mL of IL2 in the presence of 0.1 μg/mL rapamycin for a week. Cells were replaced with new culture with anti-CD3 (OKT3, eBiosciences) plate bound at 0.5 μg/mL and soluble 0.5 μg/mL anti-CD28 (clone 28.2, eBiosciences) with 100 U/mL of IL2 and 0.1 μg/mL rapamycin.
Induction of Treg cells was assayed using the alamar blue cell viability assay in the presence of anti-CD4×IL2 Fc-fusions or control antibodies. Results are shown in
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