Cells were washed with FACS buffer (HBSS containing 0.2% BSA and 0.05% Azide), treated with anti-FcR (24G2), and then stained with specific antibodies. Anti–mouse CD4, CD8, PD-1, CXCR5, B220, CD19, Fas, GL7, CD38, CD11c, B7.1, B7.2, CD40, CXCR4, CD83, TCR-Vα2, TCR-Vβ5, CD45.1, and CD45.2 antibodies were purchased from BD Biosciences. NP-PE was purchased from Biosearch Technologies. Propidium iodide was purchased from Sigma-Aldrich. For intracellular cytokine staining, splenic CD4+ T cells were stimulated with PMA and ionomycin for 2 h, and cells were fixed and permeabilized with the BD Fix/Perm kit (BD Biosciences) according to the manufacturer’s instructions and then stained with anti–IFN-γ (BD Biosciences) and isotype control antibody for 30 min. Data were collected with a FACS Calibur II, FACS LSR II, FACS Fortessa, or FACS Aria III flow cytometer (BD Biosciences) and analyzed with FlowJo software.
Tcr vα2
Manufactured by BD
Sourced in United Kingdom
TCR-Vα2 is a laboratory instrument used for the detection and analysis of T cell receptor alpha variable 2 (TCR-Vα2) expression. It is a specialized tool designed to assist researchers in the study of T cell receptor diversity and function.
Automatically generated - may contain errors
Lab products found in correlation
Facs calibur 2, by BD (1 mentions)
Anti ifn γ, by BD (1 mentions)
Fix perm kit, by BD (1 mentions)
Np pe, by Biosearch Technologies (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Facsaria 3, by BD (1 mentions)
Facs fortessa, by BD (1 mentions)
Facs lsr 2, by BD (1 mentions)
Cxcr4, by BD (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Lsr 2, by BD (1 mentions)
F4 80, by BD (1 mentions)
γδtcr gl3, by BD (1 mentions)
Isotype matched control mab, by BD (1 mentions)
Mpdca 1 bst2, by Miltenyi Biotec (1 mentions)
Ova257 264 peptide, by BD (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
Golgiplug, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
4 protocols using tcr vα2
1
Multiparameter Flow Cytometry Analysis
2
Monitoring T cell migration with CFSE and CTV
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Methods of Carboxyfluorescein succinimidyl ester (CFSE) and Cell Trace Violet (CTV) (Molecular Probes, Eugene, OR, USA) labelling to monitor OT-I and OT-II T cell migration in vivo were performed as published [9 (link),21 (link),32 (link),33 (link)]. Cells were labelled before injection, and recipient mice were euthanised at different time points after transfer for flow cytometric analysis. Splenocytes and LN cells were obtained as above. Islets were isolated with a standard collagenase protocol [13 (link)]. Isolated islets containing infiltrated lymphocytes were cultured with RPMI1640/10%FCS overnight in a 37 °C incubator to enable CD4 and CD8 T cell egress and recovery. Lymphocyte subsets and macrophages were identified using fluorescent antibodies for CD4, CD8, CD11b, CD45.1, CD45.2, F4/80, TCR Vα2, and 7AAD (BD). Heparanase-1 was detected with the Hpa 3/17 mAb supplied by Abcam, Cambridge, UK. Data were acquired on a BD LSR II and analysed using FlowJo software (Treestar, Becton Dickinson, Franklin Lakes, NJ, USA).
3
Flow Cytometric Phenotyping and Cytokine Analysis
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Cells were stained with fluorescein-conjugated mAbs to mouse CD3α (145-2C11), CD4 (RM4–5), CD8α (53–6.7), CD11c (HL3), CD40 (3/23), CD44 (IM7), CD45.1 (A20), CD62L (MEL-14), CD80 (16-10A1), CD86 (GL1), CD154 (MR1), I-A/I-E (M5/114.15.2), B220 (RA3-6B2), Vα2 TCR (B20.1), γδTCR (GL3), IFN-γ (XMG1.2), IL-17A (TC11-18H10), isotype-matched control mAb (BD Biosciences), CD209b (22D1), CD253/TRAIL (N2B2) (eBioscience), CD11b (M1/70), CD178/FasL (MFL3), F4/80 (BM8), Ly6C (HK1.4), Ly6G (1A8), γδTCR (GL3) (Biolegend), mPDCA-1/BST2 (JF05-1C2.4.1) (Miltenyi Biotec), H-2Kb OVA tetramer (MBL) and Siglec-H (440c; Hycult Biotechnology). For the intracellular expression of cytokines, cells were incubated for 4 hrs with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml; Sigma-Aldrich) plus ionomycin (500 ng/ml; Sigma-Aldrich) or OVA257–264 peptide (SIINFEKL; 10 μM) plus GolgiPlug (BD Biosciences) during the final 2 hrs. Subsequently, the cells were resuspended in Fixation-Permeabilization solution (BD Cytofix/Cytoperm kit; BD Biosciences) and intracellular cytokine staining was carried out according to the manufacturer’s directions. Fluorescence staining was analyzed with a FACSCalibur flow cytometer and CELLQuest software (both from BD Biosciences).
4
Single-Cell Immunophenotyping by Flow Cytometry
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Single-cell suspensions were obtained from isolated tissue samples. Cells were washed, counted and stained with the following murine phycoerythrin, fluorescein isothiocyanate, allophycocyanin or cyanine 7 (Cy7)—conjugated antibodies: CD4, CD8, CD44, CD25, CD30, CD117, CD3, Vα2 TCR, Notch1 (BD Biosciences, Oxford, UK). After staining (30 min at 4 °C, 1:1,000), cells were washed and analysed using fluorescence-activated cell sorter (FACS) Canto or Fortessa flow cytometers (BD Biosciences). For intracellular stain, the cells were fixed in 0.01% formaldehyde for 10 min, permeabilized in 0.5% Tween 20 v/v in PBS (15 min in the dark at RT), cells were washed in PBS 0.1% Triton and re-suspended in 100 μl of 0.1% Triton in PBS, cells were stained and analysed as previously described. Alternatively, cell populations were sorted using a FACS Aria machine as detailed in ref. 11 (link).
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