Antibodies were from BD Bioscience, eBioscience, and BioLegend. The following antibodies are listed in the order of antigen, fluorophore, clone, sources, catalog number, and dilution times for the final concentrations. CD11c, FITC, N418, Biolegend, 117306, 1600; CD11c, PE, HL3, BD, 553802, 800; F4/80, APC, BM8, eBioscience, 17–4801–82, 800; CD3, PerCP Cy5.5, 17A2, Biolegend, 100218, 800; B220, BV421, RA3-6B2, Biolegend, 103240, 800; B220, Al647, RA3-6B2, Biolegend, 103226, 1600; CD8a, APC-Cy7, 53-6.7, Biolegend, 100714, 800; CD8a, APC, 53-6.7, BD, 553035, 800; PD-1, BV421, 29F.1A12, Biolegend, 135221, 800; PD-L1, BV421, 10F.9G2, Biolegend, 124315, 800; IFN-gamma, PE, XMG1.2, BD, 554412, 100; IFN-gamma, FITC, XMG1.2, eBioscience, 11-7311-82, 100; TNF-alpha, FITC, MP6-XT22, BD, 560659, 100; TNF-alpha, APC, MP6-XT22, eBioscience, 560658, 100; CD62L, FITC, MEL-14, BD, 553150; CD44, PE-Cy5, IM7, BD, 561861. Antibodies were used according to the manufacturer’s instructions, including antibody dilution. Flow cytometry was conducted on a BD LSRFortessa X-50 flow cytometer at the Core Flow Cytometry Facility of Vaccine Research Center.
Mp6 xt22
Manufactured by BD
Sourced in United Kingdom
The MP6-XT22 is a laboratory equipment designed for general use in scientific and research settings. It is a multipurpose device that can perform various tasks as required by the user. The core function of the MP6-XT22 is to assist in the execution of laboratory procedures and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Golgiplug, by BD (3 mentions)
Cytofix cytoperm, by BD (3 mentions)
Lsrfortessa x 50 flow cytometer, by BD (1 mentions)
Mp6 xt22, by Thermo Fisher Scientific (1 mentions)
Mel 14, by BD (1 mentions)
B220 bv421 ra3 6b2, by BioLegend (1 mentions)
Bcl 10c4, by BioLegend (1 mentions)
Dan11mag, by Thermo Fisher Scientific (1 mentions)
Thr202 tyr204 d13.14.4e, by Cell Signaling Technology (1 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions)
Golgistop, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Cytofix cytoperm buffer, by BD (1 mentions)
Fcap array v3, by BD (1 mentions)
Cytometric bead array mouse th1 th2 th17 cytokine kit, by BD (1 mentions)
Golgistop, by Merck Group (1 mentions)
Il 4 11b11, by BioLegend (1 mentions)
Il 17 tc11 18h10, by BD (1 mentions)
Cd8 53 6, by BioLegend (1 mentions)
Cd45 30 f11, by BioLegend (1 mentions)
9 protocols using mp6 xt22
1
Multiparametric Flow Cytometry Immunophenotyping
2
Intracellular Cytokine and Protein Staining
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For intracellular cytokine staining (ICS), cells stimulated with indicated stimuli in the presence of GolgiStop (BD Biosciences) were stained for cell-surface markers, fixed and permeabilized using Cytofix/Cytoperm buffer (BD Biosciences) and then stained with fluorochrome-conjugated Abs to IFN-γ (XMG1.2; eBioscience; 1:300) and TNF-α (MP6-XT22; BD Biosciences; 1:300) using Perm/Wash buffer (BD Biosciences). The same ICS protocol was used for analysing Bcl-2 and CD107a expression with fluorochrome-conjugated Abs to Bcl-2 (BCL/10C4; Biolegend; 1:200) and CD107a (1D4B; eBioscience; 1:300). Intracellular staining for T-bet and Eomes expression was performed with Foxp3 Staining Buffer Set (eBioscience) according to the manufacturer's instructions using fluorochrome-conjugated Abs to T-bet (4B10; 1:200) and Eomes (Dan11mag; all from eBioscience; 1:200). For intracellular staining for p-ERK, B6 SP cells treated with indicated stimuli were fixed with 2% paraformaldehyde at RT for 15 min, followed by permeabilization with ice-cold 90% methanol for 20 min on ice. After a washing step, cells were blocked with PBS containing 2% FBS and incubated with fluorochrome-conjugated Ab to p-ERK (Thr202/Tyr204; D13.14.4E; Cell Signaling Technology; 1:100), followed by repeated washes and continued incubation for 15 min on ice with fluorochrome-conjugated Abs to cell-surface markers.
3
Characterization of Th1/Th2/Th17 Cytokines and Immune Cell Populations
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To label the Th1 and Th2 cytokines in serum, we used a Cytometric Bead Array Mouse Th1/Th2/Th17 Cytokine kit (BD Biosciences) following the manufacturer's instructions and using a FACS Canto II (BD Biosciences). To characterize the immune cell populations, single cell suspensions of lymphocytes were prepared from large intestine lamina propria
34 , 35 . Lymphocytes were stimulated for 4 h using ionomycin, phorbol myristate acetate (Sigma), and 1 μg/ml Golgistop (Sigma). Prior to flow cytometry, lymphocytes were stained with fluorescent conjugated antibodies against CD4 (RM‐4‐5, 1/800), Ifng (XMG1.2, 1/200), IL‐13 (eBIO 13a, 1/200), TCRa/b (MR5.2, 1/200) (all Fisher Scientific), CD45 (30‐F11, 1/200), CD8 (53‐6.7, 1/200), IL‐4 (11B11, 1/200) (all Biolegend, London, UK), IL‐17 (TC11‐18H10.1, 1/100) and TNF (MP6‐XT22, 1/200) (both BD Biosciences), and a live/dead marker. Data were analysed using FCAP Array v3.0 (BD Biosciences) and FlowJo (FlowJo LLC, Ashland, OR, USA) software. Statistical analysis was performed using three to five animals per genotype and three independent experiments. Significance was calculated using a three‐way full factorial fit model and a joint F‐test to assess the effects of genotype, treatment, and experiment day on the cytokine response
36 .
4
T Cell Cytokine Profiling in EAE
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Analysis of T cell responses was performed as previously detailed (Basler et al, 2009 (link)). Briefly, lymph node cells or mononucleated cells purified from brain or spinal cord were incubated in round-bottom 96-well plates with 10 μg/ml MOG35–55 peptide in 100 μl IMDM 10% plus brefeldin A (10 μg/ml) for 5–6 h at 37°C. The first 2 h of stimulation were performed in the absence of brefeldin A. The staining, fixation, and permeabilization of the cells were performed exactly as detailed previously (Basler & Groettrup, 2007 (link)). Antibodies to CD4 (clone GK1.5), GM-CSF (clone MP1-22E9), and IL-17A (clone eBio17B7) were obtained from eBioscience and antibodies to IFN-γ (clone AN-18) and TNF-α (MP6-XT22) were purchased from BD Biosciences.
5
Intracellular TNF Expression in Stimulated Thymocytes
6
Intracellular Cytokine and Transcription Factor Analysis
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For intracellular detection of in vitro cytokine and transcription factor production, cells were plated at a density of 2 × 106 ml in RPMI1640 (Gibco) containing 10% FBS, L-glutamine, and 2-mercaptoethanol. Cells were stimulated for 4-6 hours using either 1 μg/ml each, plate-bound anti-CD3ε f(ab′)2 (2C11, BioXCell), and anti-CD28 (37.51, BioXCell) in the presence of GolgiStop (3 mM monensin, BD), or Cell Stimulation Cocktail plus protein transport inhibitor (50 ng/ml phorbol 12-myristate 13-acetate (PMA), 1.34 mM ionomycin, 5.3 mM brefeldin A, 1 mM monensin) according to manufacturer guidelines (eBio). After surface staining, cells were fixed and permeabilized according to manufacturer guidelines (BD). Subsequent intracellular staining was performed with anti-TNFα (BD, MP6-XT22). Foxp3 (Fjk-16s, eBio) was detected in lymphocytes ex vivo following fixation and permeabilization according to manufacturer guidelines (eBio).
7
Intracellular TNF Expression in Stimulated Thymocytes
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Total thymocytes were stimulated with plate-bound anti-CD3 plus anti-CD28 in the presence of GolgiPlug (BD Bioscience). 4 h later, cells were treated with Cytofix/Cytoperm (BD Bioscience) for 30 min on ice after surface staining, and then stained for intracellular TNF (MP6-XT22, BD Bioscience).
8
Thymocyte TNFα Production Assay
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TNFα production in freshly isolated thymocytes was performed as described 28 . Briefly, 5 × 106 thymocytes were cultured with plate‐bound anti‐CD3 (10 ug/ml, clone 145‐2C11, eBioscience) and anti‐CD28 (20 ug/ml, clone 37.51, eBioscience) for 6 hours in GolgiPlug (BD Bioscience). Cells were stained with antibodies described below to detect SM, M1 and M2 CD4+ cells, fixed using BD Cytofix/Cytoperm (BD Bioscience) for 30 min on ice, and TNFα production was detected by flow cytometry using an anti‐TNFα antibody (MP6‐XT22, BD Bioscience).
9
Flow Cytometry Immunophenotyping Protocol
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