To investigate whether CXCR5 expression and antigen presentation function of γδ T cells were involved in the ability of γδ T cells to induce Tfh cell differentiation in vivo, γδ T cells from spleen and lymph nodes of WT C57BL/6J, CXCR5−/− and MHC-II−/− mice were first enriched using TCRγδ isolation kit (all from Miltenyi Biotec) and 5 × 105 sorted γδ T cells were intravenously transferred into TCRδ−/− mice. Three days after transfer, mice were immunized with CFA and 7 days later draining (inguinal) lymph nodes were collected for FACS analysis. To investigate the involvement of Wnt ligands secretion by γδ T cells on their ability to induce Tfh cell differentiation, WT C57BL/6J mice were treated with either 10 mg/kg of Wnt-C59 (Tocris) or vehicle (3.5% DMSO in PBS) intraperitoneally and 1 day later, animals were sacrificed and spleens and lymph nodes collected. γδ T cells were first enriched using TCRγδ isolation kit (all from Miltenyi Biotec) and 5 × 105 sorted γδ T cells were intravenously transferred into TCRδ−/− mice. One day after transfer, mice were immunized with CFA and 7 days later draining (inguinal) lymph nodes were collected for FACS analysis.
Tcrγδ isolation kit
Manufactured by Miltenyi Biotec
Sourced in Germany
The TCRγδ isolation kit is a laboratory product designed to isolate and enrich γδ T cells from a heterogeneous cell population. It provides a reliable and efficient method for the separation and purification of this specific T cell subset.
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Lab products found in correlation
Celltrace violet dye, by Thermo Fisher Scientific (4 mentions)
Cd11c microbeads, by Miltenyi Biotec (4 mentions)
Af488 conjugated anti cd11c n418, by BioLegend (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Wnt c59, by Bio-Techne (2 mentions)
Cd4 microbeads, by Miltenyi Biotec (2 mentions)
Mojosort mouse cd3 t cell isolation kit, by BioLegend (1 mentions)
Pd l1 fc chimera protein, by R&D Systems (1 mentions)
Anti icos clone c398.4a, by BioLegend (1 mentions)
Il 23, by R&D Systems (1 mentions)
Ova protein, by Merck Group (1 mentions)
Ova323 339 peptide, by InvivoGen (1 mentions)
Pd l1 fc, by R&D Systems (1 mentions)
10 protocols using tcrγδ isolation kit
1
Investigating γδ T Cells' Tfh Induction
2
Isolation and Stimulation of T Cells
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CD3+ T cells from lung single-cell suspensions (1 × 107 cells) harvested from Cre negative KB1P FVB/n or Tcrb−/− mice on C57BL/6J background were isolated by negative selection using the MojoSort mouse CD3+ T-cell Isolation kit (#480031; Biolegend) according to the manufacturer’s instructions. γδ T cell isolation was performed by TCRγδ isolation kit (#130-092-125; Miltenyi) according to the manufacturer’s instructions. Cells after enrichment were counted with a hemocytometer and plated at a density of 1 × 106 CD3+ T cells/ml or 2–3.5 × 105 γδ T cells/ml in round-bottomed 96-well plates in IMDM medium, 10% FCS, 2 mM L-glutamine, 10,000 U/ml penicillin/streptomycin, and 50 μM β-mercaptoethanol. Where indicated, wells were coated with 6 μg PD-L1-Fc chimera protein (R&D Systems) or anti-ICOS (clone C398.4A; Biolegend) in PBS overnight at 4°C. T cells were cultured in the presence of these antibodies for 3, 15, or 24 h as indicated. Cells were stimulated for last 30 min of incubation with 2.5 ng/ml recombinant IL-1β (ImmunoTools) and IL-23 (R&D Systems).
3
Adoptive Transfer of TCRγδ+LAP+ Cells
4
Isolation and Functional Characterization of Dendritic and T Cell Subsets
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CD11c+ dendritic cells and TCRγδ+CXCR5+, TCRγδ+CXCR5− cells were first enriched using CD11c microbeads or TCRγδ isolation kit (all from Miltenyi Biotec) and sorted. For TCRγδ sorting, AF488-conjugated anti-CD11c (N418; 1:100; Biolegend) was used to exclude any remaining CD11c+ cells in order to prevent dendritic cell contamination. For uptake assay, TCRγδ+CXCR5−, TCRγδ+CXCR5+ and CD11c+ dendritic cells were incubated for 3 h at 37 °C with 50 μg ml−1 of ovalbumin (OVA) coupled to Alexa Fluor 488 (Invitrogen) in a 96-well round-bottom plate. After incubation, cells were collected, thoroughly washed and analyzed by flow cytometry. For in vitro presentation assay, cells were first incubated overnight at 37 °C with 50 μg ml−1 of either OVA323–339 peptide (Invivogen), OVA protein (Sigma) or medium only (unloaded cells as control) in a 96-well round-bottom plate. On the next day, cells were thoroughly washed and incubated at 1:2 ratio (antigen-presenting cells:responder cells) with sorted naïve (CD4+CD62L+CD44−Foxp3−) cells from OT-II-Foxp3-GFP mice previously stained with CellTrace Violet dye (Invitrogen) for 4 days. Proliferation was then analyzed by flow cytometry.
5
Adoptive Transfer of TCRγδ+ Cells
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Pooled cells from spleen and PPs or thymus of C57BL/6 mice were first enriched using CD4 microbeads and TCRγδ isolation kit (both from Miltenyi Biotec), as described above and then sorted. The purity of each population was >98% as analysed by flow cytometry. To evaluate the immunomodulatory effect of TCRγδ+LAP+ cells on DSS-induced colitis model, we transferred 1 × 105 TCRγδ+LAP− or TCRγδ+LAP+ cells per animal intravenously. For the CD4+CD45RBhigh cell transfer-induced colitis model, we transferred 5 × 105 CD4+CD45RBhigh cells per animal intraperitoneally and 2.5 × 105 TCRγδ+LAP− or TCRγδ+LAP+ cells per animal intravenously. For CD45.2+TCRγδ+CD27+ cell transfer to CD45.1 congenic mice, 1 × 106 cells per mouse intravenously were used. Intravenously and intraperitoneally, cell transfers were performed in 100 and 500 μl of phosphate-buffered saline (PBS), respectively.
6
Modulation of γδ T Cell IL-17 by PD-1/PD-L1
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Standard laboratory protocols were used to prepare the splenocytes and isolate the γδ T cells by MACS using a TCRγδ isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer’s instruction. We diluted the anti-PD-1 antibody (eBioscience, San Diego, CA, USA) into 10 and 20 µg/mL, the PD-L1-Fc fusion protein (PD-L1-Fc, R&D Systems, Minneapolis, MN, USA) into 5 and 10 µg/mL with PBS respectively. According to the report (21 (link)), The anti-PD-1 antibody and PD-L1-Fc were coated in 96-well plates overnight at 4 °C. In each well, 2×105 MACS-purified γδ T cells (purity was greater than 90.0%) were added, and cultured in RPMI 1640 medium containing 10% FBS with 5 µg/mL anti-CD3 antibody for 48 hours. In control group, the cells were cultured in the plate precoated with rat IgG. IL-17A-producing γδ T cells were examined by flow cytometry and the level of IL-17A in culture supernatant was determined by ELISA.
7
Antigen Presentation and T Cell Activation
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CD11c+ dendritic cells, TCRγδ+CXCR5+ and TCRγδ+CXCR5− cells from WT mice were first enriched using CD11c microbeads or TCRγδ isolation kit (all from Miltenyi Biotec) and sorted. For TCRγδ sorting, AF488-conjugated anti-CD11c (N418; 1:100; Biolegend) was used to exclude any remaining CD11c+ cells in order to prevent dendritic cell contamination. Cells were then incubated overnight at 37 °C with 50 μg ml−1 of either OVA323–339 peptide, OVA protein or medium only (unloaded cells as control) or 50 μg ml−1 of OVA323–339 peptide plus either 1 μM of the porcupine (PORCN) inhibitor (Wnt-C59, Tocris) or 20 μg ml−1 of anti-Wnt8b monoclonal antibody (LSBio) in a 96-well round-bottom plate. On the next day, cells were thoroughly washed and incubated at 1:2 ratio (antigen-presenting cells:responder cells) with sorted naïve (CD4+CD62L+CD44−Foxp3−) cells from OT-II-Foxp3-GFP mice previously stained with CellTrace Violet dye (Invitrogen) for 4 days. CXCR5 expression on CD4 T cells was then analyzed by flow cytometry.
8
Dendritic Cells and TCRγδ+ Cell Uptake and Presentation
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CD103+CD11c+, CD103-CD11c+ dendritic cells and TCRγδ+LAP+, TCRγδ+LAP− cells were first enriched using CD11c microbeads or TCRγδ isolation kit (all from Miltenyi Biotec) and sorted. For uptake assay, TCRγδ+LAP− and TCRγδ+LAP+ cells were incubated for 3 h at 37 °C with 50 μg ml−1 of ovalbumin (OVA) coupled to Alexa Fluor 488 (Invitrogen) in a 96-well round-bottom plate. After incubation, cells were collected, thoroughly washed and analysed by flow cytometry. For presentation assay, sorted CD103-CD11c+, CD103+CD11c+ dendritic cells and TCRγδ+LAP+, TCRγδ+LAP− cells (from WT or MHC-II−/− mice) were first incubated overnight at 37 °C with 50 μg ml−1 of OVA323–339 peptide or medium only (unloaded cells as control) in a 96-well round-bottom plate. Next day, the cells were thoroughly washed and incubated at 1:1 ratio with sorted naive (CD4+CD62L+CD44-Foxp3−) cells from OT-IIxFoxp3-GFP mice previously stained with CellTrace Violet dye (Invitrogen) for 4 days. Proliferation and Foxp3 induction were then analysed by flow cytometry. In some experiments, purified anti-LAP mAb (TW7–16B4) was used to study the involvement of LAP in the Foxp3 induction by TCRγδ+LAP+ cells at a concentration of 30 μg ml−1.
9
Dendritic Cells and T Cell Antigen Presentation
10
CXCR5 Modulation by γδ T Cell Subsets
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TCRγδ+CXCR5+ and TCRγδ+CXCR5− cells from WT mice were first enriched using TCRγδ isolation kit (all from Miltenyi Biotec) and sorted. AF488-conjugated anti-CD11c (N418; 1:100; Biolegend) was used to exclude any remaining CD11c+ cells in order to prevent dendritic cell contamination. Cells were then stimulated with plate-bound anti-CD3 (145–2C11; Biolegend) and anti-CD28 (PV-1; Bioxcell), both at 2 μg ml−1, for 3 days at 37 °C in a 96-well round-bottom plate. Wnt-C59 at 1 μM was added to some wells to prevent Wnt ligand release. At the 3rd day of culture, supernatants were collected and 100 μl added to naïve (CD4+CD62L+CD44−Foxp3−) cells in a 96-well round-bottom plate and stimulated with plate-bound anti-CD3 and anti-CD28 (both at 2 μg ml−1) at 37 °C. Three days later, CXCR5 expression on CD4 T cells was analyzed by flow cytometry.
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