Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density gradient centrifugation (Lymphoprep, STEMCELL Technologies, Cambridge, UK). Isolated PBMCs were preserved in FCS +10% DMSO and stored in liquid nitrogen prior to use. Cryopreserved PBMCs were thawed rapidly at 37°C and washed with 1x PBS, T cells were isolated by pan T cell isolation kit (Miltenyi Biotec) as per the protocol. T cells were resuspended in RPMI 1640 medium supplemented with 10mM glucose, 0.1mM non-essential amino acids, 10mM HEPES buffer, 1mM sodium-pyruvate, 50 IU/mL of streptomycin and 50 IU/mL penicillin (all Sigma-Aldrich) and 10% FCS. T cells were kept in a 96 well plate at 200,000 cells/ well in an incubator at 37°C, 5% CO2. On Day 1, T cells were stimulated with plate-bound anti-CD3 (OKT3 clone, 1 μg/mL) and anti-CD28 (CD28.2 clone, 0.5 μg/mL) (BioLegend) overnight with 20 IU/mL of interleukin (IL)-2. On day 2, T cells were removed from stimulation to rest. On day 5, cells were replenished with fresh media and 20 IU/mL IL-2. On day 5 and day 6, 5μg EV in 25 ul 1X PBS were added per well. On day 7 cells were restimulated with plate-bound anti-CD3 and anti-CD28 (BioLegend) and 1 μg/mL Brefeldin A (BFA; Biolegend) was added per well. Cells were analysed on day 8.
Anti cd3 and anti cd28
Manufactured by BioLegend
Sourced in United States
Anti-CD3 and anti-CD28 are antibodies used in cell culture research to activate and expand T cells. Anti-CD3 binds to the CD3 receptor on T cells, while anti-CD28 binds to the CD28 receptor, providing co-stimulatory signals necessary for T cell activation and proliferation. These antibodies are commonly used to stimulate and study T cell responses in vitro.
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Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Anti cd21 cd35, by Miltenyi Biotec (2 mentions)
Anti cd45r, by Miltenyi Biotec (2 mentions)
Anti cd49, by Miltenyi Biotec (2 mentions)
Il 33, by Thermo Fisher Scientific (2 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (2 mentions)
Anti cd11b, by Miltenyi Biotec (2 mentions)
Anti ter 119, by Miltenyi Biotec (2 mentions)
Anti cd3, by Miltenyi Biotec (2 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Anti cd28 cd28.2 clone, by BioLegend (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Lymphoprep, by STEMCELL (1 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (1 mentions)
Foxp3 transcription factor staining set, by Thermo Fisher Scientific (1 mentions)
Fixation permeabilization solution kit, by BD (1 mentions)
Golgiplug protein transport inhibitor, by BD (1 mentions)
9 protocols using anti cd3 and anti cd28
1
Isolation and Activation of Human T Cells
2
Flow Cytometry for Immune Profiling
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To block Fc receptors, human samples were incubated in 5% normal rat serum and murine samples were incubated in TruStain FcX (BioLegend) and normal rat serum then stained using antibodies listed in online supplemental table 4 . Intracellular staining of murine Foxp3 was performed according to manufacturer instructions using a Foxp3/Transcription factor staining set (ThermoFisher). For murine IFNγ, TNF⍺ and perforin intracellular staining, bulk tumor cells were ex vivo stimulated for 4 hours with plate-bound, purified anti-CD3 and anti-CD28 (BioLegend). GolgiPlug protein transport inhibitor (BD Biosciences) was added for the final 2 hours. Cells were harvested and stained using BD Biosciences Fixation/Permeabilization Solution Kit. Results were obtained using an Attune NxT (ThermoFisher) or BD LSR II (BD Biosciences) flow cytometer and analyzed with FlowJo Software. Dead cells were excluded via Zombie Aqua or Green Fixable Viability Dye (BioLegend). Fluorescence minus one controls were used to objectively determine gate boundaries for positive events.
3
Stimulating T Cell Activation via DCs
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DCs were infected with SL3261 murine cyclic GMP–AMP synthetase (mcGAS) or SL3261 mcGASAA for 30 min, spun down, and the medium was replaced with RPMI-1640 supplemented with 10% FCS and 100 µg/mL gentamycin. Next, the stimuli LPS or cGAMP (10 µg/mL, Invitrogen) were added to the corresponding wells. The cGAMPs were incubated with transfection reagent Lyovec (Invivogen) according to manufacturer’s instructions to ensure intracellular delivery of the cGAMPs prior to adding to DCs. After 16-hour incubation, DCs were cocultured with allogeneic PBLs in Iscove’s Modified Dulbecco’s Media (Gibco) supplemented with 10% FCS and 1% penicillin/streptomycin, using a 1:8 ratio. As a positive control, T cells were stimulated with plate-bound anti-CD3 and anti-CD28 (BioLegend). After 3 days, IL-2 (20 U/mL, Chiron) was added to the cocultures. After 6 days, PBLs were harvested, plated in 96-well plates, and incubated for 4 hours with brefeldin A (10 µg/mL, Sigma), after which they were analyzed for cytokine production by flow cytometry as described further.
4
Coculture of Cancer and Immune Cells
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Cancer cells were seeded in a 24-well plate, and naïve immune cells processed from spleens of wild-type 129sv mice were added at 1:6–1:8 ratio. All cocultures were supplemented with 5 μg/mL anti-CD3 and anti-CD28 (BioLegend or BioXcell) antibodies and 50 μM β-mercaptoethanol. Cells were cocultured and induced with doxycycline for 96 h at 37°C with 5% CO2. For conditioned media assays, conditioned media was collected after 72 h of doxycycline induction of cancer cells, centrifuged at 1500 rpm to remove potential detached cancer cells, and then added to naïve immune cells and cultured for 72–96 h at 37°C with 5% CO2.
5
Isolation and Activation of Murine T Cells
6
Antibody Panel for Epigenetic Markers
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The mouse antibody anti-H3K9me2/3, and the rabbit antibodies anti-H3K9ac, -H3K4me3, -H4K20me3, -H3 and -G9a were from Cell Signaling (Beverly, MA, USA). Rabbit anti-GLP was from Thermo- Scientific (Waltham, MA, USA). Mouse anti-lamin B1 (for the HMT experiment) was from Santa Cruz Biotechnology (Dallas, TX, USA) and rabbit anti-lamin B1 was from Abcam (Cambridge, UK). The rabbit antibody against suv39h1 was from Abcam and the mouse anti-β-tubulin was from Sigma-Aldrich (St. Louis, MO, USA). Anti-CD3 and anti-CD28 were from Biolegends (San Diego, CA, USA). 12G10 (anti-β1 activator Ab), 9EG7 (anti-β1 activator Ab) and mab13 (anti-β1 blocking Ab) were kindly provided by Martin Humphries (University of Manchester, UK). VCAM1 was obtained from Martin Humphries and Peprotech. Fibronectin fragment FN-H50 and FN-H120 were kindly provided by Martin Humphries. Fluorescence-conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, PA, USA). ICAM1, IL-4, CXCL12 and CCL19 were from Peprotech (Rocky Hill, NJ, USA). BIX01294, chaetocin, actinomycin were from Sigma-Aldrich. Hoechst 33342 was from Invitrogen (Grand Island, NY, USA). CFSE and Cell Tracer Far Red were from Life Technologies (Paisley, UK).
7
PBMC Proliferation Assay with PD-L1 Inhibition
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Peripheral Blood Mononuclear Cells (PBMCs) were isolated from heparin-anticoagulated whole blood using a Ficoll-Paque PLUS density gradient (GE Healthcare Biosciences, Sweden), and cryopreserved in medium containing 90% fetal calf serum (HyClone) and 10% dimethyl sulfoxide (Corning, USA). Prior to the experiment, cells were thawed and rested overnight in a 37°C CO2 incubator. For proliferation assays, Carboxyfluorescein succinimidyl ester (CFSE) (Biolegend USA) labeled PBMC were stimulated for 4 days with plate bound 1 μg/ml anti-CD3 and anti-CD28 (Biolegend, USA) antibodies and 10ug/ml of PD-L1 Ig or Control Ig protein (Biolegend, USA) with varying concentrations of Kynurenine (Tocris, USA). Four days later, cells were stained with CD3-APCCy7 antibody (Biolegend, USA) and acquired in BD FACSAria instrument. Results were analyzed in Flow Jo software. Supernatants were analyzed for IFNγ using the kit, Human IFN gamma ELISA Ready-SET-Go according to the manufacture's instruction.
8
Isolation and Culture of Murine CD8+ T Cells and ILC2s
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Murine CD8 + T cells were isolated from tumor tissue using the tumor in ltration CD8 + T-cell magnetic bead sorting kit (Miltenyi Biotec, Belgish-Gladbach, Germany). Cell purity was con rmed by ow cytometry (96%). Sorted CD8 + T cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Gibco, NY, USA), 2 µg/ml anti-CD3 and anti-CD28 (Biolegend), 10 U/ml penicillin and 10 U/ml streptomycin (Gibco).
ILC2s were sorted from murine spleen by a combination of microbeads and ow cytometry. Brie y, the murine splenic cell suspension was incubated with biotin-binding antibodies (anti-CD21/CD35, anti-Ter-119, anti-CD45R, anti-CD3, anti-CD11b, and anti-CD49; Miltenyi Biotec) to enrich lineage negative cells. The enriched lineage negative cells were further stained with FITC-lineage, PE-CD90.2 and APC-KLRG1 (Biolegend) and then sorted by ow cytometry. The purity of the sorted ILC2s was approximately 90% and the sorted ILC2s were cultured in RPMI 1640 medium supplemented with 10% FBS, 10 ng/ml IL-7, 10 ng/ml IL-33 (Peprotech), 10 U/ml penicillin and 10 U/ml streptomycin.
ILC2s were sorted from murine spleen by a combination of microbeads and ow cytometry. Brie y, the murine splenic cell suspension was incubated with biotin-binding antibodies (anti-CD21/CD35, anti-Ter-119, anti-CD45R, anti-CD3, anti-CD11b, and anti-CD49; Miltenyi Biotec) to enrich lineage negative cells. The enriched lineage negative cells were further stained with FITC-lineage, PE-CD90.2 and APC-KLRG1 (Biolegend) and then sorted by ow cytometry. The purity of the sorted ILC2s was approximately 90% and the sorted ILC2s were cultured in RPMI 1640 medium supplemented with 10% FBS, 10 ng/ml IL-7, 10 ng/ml IL-33 (Peprotech), 10 U/ml penicillin and 10 U/ml streptomycin.
9
Isolation and Expansion of Murine CD8+ T Cells and ILC2s
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Murine CD8 + T cells were isolated from tumor tissue using the tumor infiltration CD8 + T-cell magnetic bead sorting kit (Miltenyi Biotec, Belgish-Gladbach, Germany). Cell purity was confirmed by flow cytometry (96%). Sorted CD8 + T cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Gibco, NY, USA), 2 µg/ml anti-CD3 and anti-CD28 (Biolegend), 10 U/ml penicillin and 10 U/ml streptomycin (Gibco).
ILC2s were sorted from murine spleen by a combination of microbeads and flow cytometry. Briefly, the murine splenic cell suspension was incubated with biotin-binding antibodies (anti-CD21/CD35, anti-Ter-119, anti-CD45R, anti-CD3, anti-CD11b, and anti-CD49; Miltenyi Biotec) to enrich lineage negative cells. The enriched lineage negative cells were further stained with FITC-lineage, PE-CD90.2 and APC-KLRG1 (Biolegend) and then sorted by flow cytometry. The purity of the sorted ILC2s was approximately 90% and the sorted ILC2s were cultured in RPMI 1640 medium supplemented with 10% FBS, 10 ng/ml IL-7, 10 ng/ml IL-33 (Peprotech), 10 U/ml penicillin and 10 U/ml streptomycin.
ILC2s were sorted from murine spleen by a combination of microbeads and flow cytometry. Briefly, the murine splenic cell suspension was incubated with biotin-binding antibodies (anti-CD21/CD35, anti-Ter-119, anti-CD45R, anti-CD3, anti-CD11b, and anti-CD49; Miltenyi Biotec) to enrich lineage negative cells. The enriched lineage negative cells were further stained with FITC-lineage, PE-CD90.2 and APC-KLRG1 (Biolegend) and then sorted by flow cytometry. The purity of the sorted ILC2s was approximately 90% and the sorted ILC2s were cultured in RPMI 1640 medium supplemented with 10% FBS, 10 ng/ml IL-7, 10 ng/ml IL-33 (Peprotech), 10 U/ml penicillin and 10 U/ml streptomycin.
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