Fifty to 70 ml of blood was taken by venous puncture. Peripheral blood mononuclear cells were isolated from heparinized blood by Ficoll-Paque (GE Healthcare) purification. Briefly, blood was diluted 1:1 with sodium chloride and tubes were centrifuged at 400g for 30 minutes with the brake off. Cells were harvested from the interface of the Ficoll layer, and were washed and enumerated. Monocytes were isolated using anti-CD14 microbeads, according to the manufacturer’s instruction (Miltenyi Biotec, San Diego, CA, USA). Cells were cultured in RPMI 1640 with 10% human serum and L-glutamine, pen/strep, non-essential amino acids, sodium pyruvate, 2-ME, 40 ng/ml of recombinant human IL-4 and 40 ng/ml of recombinant human GM-CSF (both from R&D Systems). Cytokines were replenished on days 3 and 6, and cells were used on day 7. For some experiments, CD4+ T cells were isolated from the CD14- fraction using the T cell isolation II kit (Miltenyi Biotec). Cells were then cultured with MoDCs in the presence or absence or RSV. At 48 hours, RNA was extracted and message levels of IFN-γ, IL-5 and IL-13 were determined by qPCR.
T cell isolation kit 2
Manufactured by Miltenyi Biotec
The T cell isolation kit II is a laboratory equipment product designed for the isolation of T cells from biological samples. It provides a simple and efficient method to separate T cells from other cell types, enabling further analysis or experimentation with the isolated T cell population.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll paque, by GE Healthcare (1 mentions)
Pen strep, by R&D Systems (1 mentions)
Recombinant human gm csf, by R&D Systems (1 mentions)
Recombinant human il 4, by R&D Systems (1 mentions)
Sodium pyruvate, by R&D Systems (1 mentions)
Anti cd14 microbeads, by Miltenyi Biotec (1 mentions)
Bio plex cytokine assay, by Bio-Rad (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Interleukin 2 (il 2), by Roche (1 mentions)
Anti pd l1, by Thermo Fisher Scientific (1 mentions)
Pd l1 cd274, by BD (1 mentions)
Apc conjugated hla dr, by BD (1 mentions)
Anti ser9 phospho gsk 3β, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti phospho nf κb p65 ser536, by Cell Signaling Technology (1 mentions)
Cd14 magnetic beads, by Miltenyi Biotec (1 mentions)
Interleukin 4 (il 4), by Miltenyi Biotec (1 mentions)
Ifn γ, by BD (1 mentions)
Gm csf, by Miltenyi Biotec (1 mentions)
Hrp conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
8 protocols using t cell isolation kit 2
1
Monocyte-Derived Dendritic Cell Generation
2
Antigen-Specific CD4+ T Cell Activation
3
Isolation and Purification of Immune Cell Subsets
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Human PBMC were isolated from heparinized blood, and splenic mononuclear cells (SMNCs) were isolated from mechanically dissociated and filtered human and murine spleens (from C57BL/6 mice) using Ficoll density gradient centrifugation (Ficoll PaquePlus; GE Healthcare, Amersham). CD19+ B cells, CD14+ monocytes and CD4+ T cells were isolated by positive magnetic selection (Miltenyi Biotec). Prior to flow sorting Tregs, untouched Pan T cells were enriched by negative selection using T-cell isolation kit II (Miltenyi biotec).
4
Isolation and Culture of Primary Cells
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Blood was acquired from the NHS blood service under ethics license REC 05/Q0401/108 (University of Manchester). Lymphocyte populations were isolated and cultured as previously described, as were 721.221 cells (33 (link)) with the following additions: T cells were isolated using T cell isolation kit II (Miltenyi Biotec); NK cells were stimulated with 200 units/ml IL-2 (Roche), and all primary cells were rested at least 4 days prior to experiments.
5
Multiparametric Immunophenotyping of DCs
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Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (mAbs) to CD86, CD1a, PD-L1 (CD274), IFN-γ (all anti-human), phycoerythrin (PE)-conjugated mAbs to CD80, CD83 and CD25, APC-conjugated HLA-DR (all anti-human), anti-human Alexa Fluor 700-CD4 were from BD Biosciences, and PE-conjugated mAb to anti-human CD40 was from Becton Dickinson. Human Foxp3-APC, anti-PD-L1 and isotype control were from eBioscience. CD14 magnetic beads, T cell isolation kit II, GM-CSF and IL-4 were from Miltenyi Biotec. Anti-β-ACTIN and anti-PGE2 antibodies were purchased from Sigma-Aldrich. Anti-SHH, anti-GLI1, anti-NUMB, anti-Ser9 phospho-GSK-3β, anti-NICD (Cleaved Notch1), anti-Ser2448 phospho mTOR, anti-Tyr458 phospho p85 and anti-Ser536 phospho NF-κB p65 were purchased from Cell Signaling Technology. Anti-COX-2 was from Calbiochem. Anti-Notch 2 intracellular domain and anti-Notch 4 -C-terminal antibodies were from Abcam. HRP conjugated anti-rabbit IgG and anti-mouse IgG was obtained from Jackson ImmunoResearch.
6
Isolation of Kidney and Splenic T Cells
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Kidney T cells were isolated using FACS. Briefly, single-cell suspension of KMNCs was preincubated with anti-CD16/CD32 Fc block (clone S17011E, BioLegend, 156604) stained in Cell Staining Buffer (BioLegend) with fluorochrome-labeled antibodies: APC-Cy7 anti-CD45 (clone 30-F11, BioLegend, 103116) and BV421 anti-TCRβ (clone H57-957, BioLegend, 109230). Live Dead Aqua (Thermo Fisher Scientific) was stained for viability assay. Live Dead Aqua–CD45+TCRβ+ cells were sorted with FACSAria II Cell Sorter (BD Biosciences). Splenic T cells were isolated from single-cell suspension of spleens using a T Cell Isolation Kit II (Miltenyi Biotec) according to the manufacturer’s guidelines.
7
OTII T cell adoptive transfer and Tfh analysis
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CD4+CD62L+ T cells from spleen, MLN, and PP of WT OTII and Pou2af1−/− OTII mice were isolated by MACS using the T Cell Isolation Kit II (Miltenyi Biotec). On the same day, CD4−/− mice received either WT or Pou2af1−/− CD4+CD62L+OTII+ T cells (8 × 106 cells per animal, i.v.). One day later, mice were immunized with OVA + alum. On day 7 after immunization, spleen, MLN, and PP were harvested and analyzed for the development of Tfh cells.
8
Allogeneic DC-Induced CD4+ T Cell Proliferation
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CD4+ T cells were negatively selected from peripheral blood mononuclear cells (PBMCs) by using the T cell isolation kit II from Miltenyi Biotec. Mixed lymphocyte reaction (MLR) was performed in 96-well U bottom plates (Nunc). 1 × 105 CD4+ T cells were incubated for 5 days in RPMI with 10% FCS together with 1 × 104 to 1 × 103 allogeneic DCs. Experiments were conducted in quadruplicate. At day 5, the proliferative response was measured by the [3H]-thymidine ([3H]-Thy, 1 µCi/mL, Amersham) incorporation test. [3H]-Thy was added for the last 8 h of incubation. Plates were then harvested (TomtecMacIII) on glass fiber filters (Perkin Elmer), and [3H]-Thy uptake was measured by liquid scintillation in a Microbeta 1450 Trimux counter (Wallac). The proliferative response is reported as a stimulation index (SI, mean cpm response/mean cpm background).
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