Naïve CD4+ T cells were positively selected from spleens and LNs using anti-CD4 microbeads (Miltenyi Biotec, Auburn, CA, USA). CD8+ cells were depleted using anti-CD8 microbeads (Miltenyi Biotec), and the remaining cells were regarded as APCs. CD4+ T cells and APCs were cultured in complete RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. For antigen-specific stimulation, isolated naïve CD4+ T cells (2 × 105) and APCs ((1 × 105) from OT-II mice were incubated with OVA323–339 peptide (0.1 μM) under Th1-polarizing conditions (10 ng/ml IL-12; BioLegend, 5 μg/ml anti-IL-4; BioLegend), Th17-polarizing conditions (1 ng/ml TGF-β1; R&D Systems, 10 ng/ml IL-6; R&D Systems, 5 μg/ml anti-IL-4; BioLegend, 5μ/ml anti–IFN-γ; BioLegend).
Anti cd4 microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany, United States, Japan
Anti-CD4 microbeads are a type of magnetic beads used for the isolation and enrichment of CD4+ T cells from biological samples. These beads are coated with antibodies specific to the CD4 surface antigen, allowing for the efficient capture and separation of CD4+ cells from a heterogeneous cell population.
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Lab products found in correlation
Anti cd8 microbeads, by Miltenyi Biotec (7 mentions)
Facsaria, by BD (6 mentions)
Anti cd3, by Thermo Fisher Scientific (4 mentions)
Anti cd28, by Thermo Fisher Scientific (4 mentions)
Facsaria cell sorter, by BD (4 mentions)
Anti pe microbeads, by Miltenyi Biotec (3 mentions)
Complete freund s adjuvant, by Merck Group (3 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (3 mentions)
Automacs, by Miltenyi Biotec (3 mentions)
Anti cd3, by BD (3 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Ls column, by Miltenyi Biotec (3 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (3 mentions)
Celltrace violet cell proliferation kit, by Thermo Fisher Scientific (3 mentions)
Interleukin 6 (il 6), by R&D Systems (2 mentions)
Hiseq 4000 pe 100 wgbs, by Illumina (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Il 1β, by R&D Systems (2 mentions)
Phytohemagglutinin (pha), by Merck Group (2 mentions)
Interleukin 2 (il 2), by BD (2 mentions)
94 protocols using anti cd4 microbeads
1
Antigen-Specific Naive CD4+ T Cell Polarization
2
Whole-Genome Bisulfite Sequencing of CD4+ T Cells
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Forty milliliters of blood were obtained from each study subject and processed fresh within 4 h of being drawn. CD4+ T cells were positively selected [anti-CD4 microbeads (Miltenyi Biotec) and auto-MACS] and their purity assessed with flow cytometry. Only samples with a purity > 95% were used for genomic DNA extraction and sequencing. The samples were processed using the in-house DNA isolation and Illumina HiSeq 4000 PE 100 WGBS workflows at the McGill University and Genome Quebec Innovation Centre. Quality control of the genetic materials was performed using fluorescence assay quantification, agarose gel electrophoresis and NanoDrop nucleic acid quantification to ensure sufficient quantity, quality and purity (Additional file 18 :).
3
Yop Translocation Quantification in CD4+ T Cells
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For in vitro analysis of Yop translocation, total CD4+ T cells were isolated from spleens and LNs of Foxp3hCD2 mice using anti-CD4-Microbeads and the autoMACS Pro separation system (Miltenyi Biotec). Subsequently, cells were co-cultured with Yptb-WT-Bla or Yptb-ΔT3SS-Bla at the indicated MOIs at 37 °C for 1 h and washed twice with RPMI medium supplemented with 50 μg/mL gentamicin to eliminate bacteria. Cells were stained for cell surface markers anti-CD4 and anti-hCD2 and labeled with CCF4-AM, using the LiveBLAzer-FRET B/G loading kit (Thermo Fisher Scientific) for 1 h at 24 °C in the presence of 1.5 mM probenecid (Sigma-Aldrich) and 50 μg/mL gentamicin. Finally, cells were harvested and analyzed by flow cytometry.
4
Assessing CD4+ T Cell Activation by BMDCs
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Bone marrow-derived dendritic cells (BMDCs) were generated from bone marrow cell cultures as previously described [43] (link). CD4+ T cells were obtained from C57BL mice, which were immunized with SIVgag antigen, and isolated using anti-CD4 microbeads (Miltenyi). The purified CD4+ T cells were co-cultured with BMDCs. CD4+ T cells were used as effector cells, and BMDCs loaded with different antigens were used as target cells. The ratios were 3∶1 or 9∶1 (E:T). After 48 h, the culture supernatants were measured using an IFN-γ ELISA kit (Dakewei). Mouse IFN-γ at concentrations of 7.8 pg/ml −500 pg/ml was used as a standard. If IFN-γ levels in the supernatants exceeded 500 pg/ml, then the supernatants were diluted and IFN-γ levels were re-measured.
5
Adoptive Transfer of MOG-Specific Th17 Cells
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B6.SJL donor mice (CD45.1+) were induced as an EAE model. Ten days later, dLNs were isolated and then restimulated with 50 μg/mL of MOG peptide and Th17 cell–polarizing factors (20 ng/mL rhIL-6, 20 ng/mL rhIL-23, 20 ng/ml IL-1β and 10 μg/mL anti-IL4, and 10 μg/mL anti–IFN-γ) to generate MOG-specific Th17 cells. After 4 days in culture, the resting cells were sorted with anti-CD4 microbeads (Miltenyi Biotec), the number of CD4+ T cells was calculated, and then 1 × 106 CD4+ T cells per mouse were intravenously injected into irradiated C57BL/6J recipient mice (4 Gy). Next, the mice were injected with 200 ng pertussis toxin in PBS on day 0 and 2 days after transfer.
6
CD4+ T cell and B cell transfer for CIA
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CD4 T cells were purified from the draining LN of WT mice or Il21r KO mice 14 days after immunization with CII/CFA using anti-CD4 microbeads and an autoMACS cell separator (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. B cells were purified from the LNs and the spleens of naïve WT mice or Il21r KO mice using PE-conjugated anti-CD19 mAb (1D3; BD Bioscience) and anti-PE microbeads (Miltenyi Biotec). The purity of both CD4+ T cells and CD19+ B cells was greater than 95 %. The purified CD4+ T cells (3 × 107/μl) and B cells (1 × 108/μl) were injected i.v. into Rag2 KO mice, which were subsequently immunized with chicken CII and CFA to induce CIA. We confirmed the presence of the transferred CD4+ T cells and CD19+ B cells in the peripheral blood of recipient RAG2 KO mice by flow cytometry prior to CII immunization.
7
Adoptive Transfer of CD4+ T Cells and CD19+ B Cells Confers Protective Immunity Against GAS Infection
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C57B/6 mice (4 to 6 weeks) were i.n. immunized with SrtA/CTB three times. 10 days after the last immunization, lymph nodes and the spleens were harvested and pooled. Single cell suspensions were made and CD4+ T cells were purified ( >93%) using anti-CD4 microbeads (Miltenyi Biotec) according to the manufacturer's protocol [15] (link). For purifying CD19+ B cells, single cell suspensions were sorted using a FACSAria sorter with FACSDiva software (BD Bioscience) after FITC-conjugated anti-CD19 (Biolegend) staining. A small volume from each sample was stained and analyzed by flow cytometry for the purity and phenotype of the cells. The purities of sorted CD4+ T cells and CD19+ B cells were 93% and 95%, respectively. Naïve female C57B/6 mice aged 4 to 6 weeks were used as recipients, and 1×107 of either CD4+ T cells or CD19+ B cells were transferred to each mouse in a volume of 200 µl by tail vein injection. 24 hr later, recipient mice were challenged i.n. with 2×108 CFUs of GAS M28 per mouse. All mice were killed 24 hr after the challenge; NALTs were harvested, and cell lysates were diluted and plated on sheep blood agar plates for CFU counts.
8
RSV-Specific T Cell Responses
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Eight days following RSV infection, CD4+ T cells and CD8+ T cells were isolated from the mouse spleens using anti-CD4 micro beads or anti-CD8 micro beads (Miltenyi Biotec, Auburn, CA, USA) in accordance with the manufacturer's instructions. CD4+ T and CD8+ T cells (2×105) were re-stimulated with heat-inactivated RSV or RSV M197-195 peptide, pulsed with irradiated APCs (3×105), and cultured for 72 hr at 37℃ in 96-well U-bottom plates (BD falcon, Durham, NC, USA). IFN-γ production in the supernatants was measured by ELISA with anti-mouse IFN-γ (eBioscience, XMG1.2) as the capture antibody and biotin-labeled anti-mouse IFN-γ (eBioscience, R4-6A2) as the secondary antibody.
9
Purification and Profiling of T Cell Subsets
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Red blood cells were lysed with ACK lysis buffer (Stem Cells Inc., Newark, CA). Single cell suspensions were prepared and Fc receptor was blocked with antibodies against CD16/ CD32 (eBioscience, San Diego, CA), followed by staining for cell surface expression of CD3 (145-2C11), CD4 (RM4-5), CD25 (PC61.5), CD44 (IM7), CD62L (MEL-14), CD103 (2E7), Icos (7E.17G9), CD127 (A7R34), CD69 (H1.2F3) (eBioscience; BD Biosciences, San Jose, CA) or intracellular staining for FOXP3 (FJK-16s) and CTLA4 (UC10-4B9). Flow cytometry data were acquired on a FACS Canto-II flow cytometer (BD Biosciences), and data were analysed using FLOWJO software (Tree Star, Ashland, OR). Sharpin +/+ ; Foxp3 gfp/gfp and Sharpin cpdm/cpdm ; Foxp3 gfp/pfg mice were isolated by positive selection with anti-CD4 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany).
10
Isolation and Characterization of Murine T-Cell Subsets
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CD4+ T cells were enriched from spleen and lymph nodes of aged female Balb/c mice by magnetic-activated cell sorting (MACS) using anti-CD4 MicroBeads and the autoMACS magnetic separation system (Miltenyi Biotec). Enriched CD4+ T cells were stimulated for 3 h with phorbol 12-myristate 13-acetate (PMA; 10 ng/ml, Sigma-Aldrich) and ionomycin (500 ng/ml, Sigma-Aldrich), followed by the labeling of IL-17A and/or IFN-γ-secreting cells using the IL-17A and IFN-γ secretion assay kit (Miltenyi Biotec) according to the manufacturer's instructions. Subsequently, the cells were stained with fluorochrome-conjugated anti-CD3 (145-2C11), CD4 (RM4-5) and CD45RB (C363.16A) antibodies. Th17 cells (CD3+CD4+CD45RBlowIL-17A+IFN-γ−), Th1 cells (CD3+CD4+CD45RBlowIL-17A−IFN-γ+), Th-mem cells (CD3+CD4+CD45RBlowIL-17A−IFN-γ−) and naive CD4+ T cells (CD3+CD4+CD45RBhighIL-17A−IFN-γ−) were sorted by fluorescence-activated cell sorting (FACS) on a FACSAria II (BD Biosciences). Purities of sorted populations ranged between 97% and 99%. Tregs were sorted from the same source as CD3+CD4+CD25high cells (anti-CD25 antibody PC61.5) and subsequently stimulated with the same concentration of PMA/ionomycin for 3 h. Treg purity was confirmed by intracellular staining for Foxp3 (FJK-16S), IL-17A (eBio17B7) and IFN-γ (XMG1.2) using the Foxp3 transcription factor staining buffer set (eBioscience).
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