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Anti mhcii fitc

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Anti-MHCII-FITC is a fluorescently labeled antibody that binds to major histocompatibility complex class II (MHC-II) proteins. It is used in flow cytometry and other immunology applications to identify and analyze cells expressing MHC-II.

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Lab products found in correlation

Anti cd86 pe, by Thermo Fisher Scientific (4 mentions) Pecy7 anti cd45, by BD (4 mentions) Anti cd11c alexa700, by Thermo Fisher Scientific (4 mentions) Penicillin streptomycin, by Merck Group (4 mentions) Anti cd11b pe cy7, by Thermo Fisher Scientific (2 mentions) Anti cd25 pe, by Thermo Fisher Scientific (2 mentions) Anti foxp3 percp, by Thermo Fisher Scientific (2 mentions) Anti cd11c apc, by Thermo Fisher Scientific (2 mentions) Collagenase, by Thermo Fisher Scientific (2 mentions) Anti f4 80 apc, by Thermo Fisher Scientific (2 mentions) Dispase, by Thermo Fisher Scientific (2 mentions) Anti cd16 32, by Thermo Fisher Scientific (2 mentions) Anti cd103 pe, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Merck Group (2 mentions) Lps free fbs, by Thermo Fisher Scientific (2 mentions) Rpmi 1640, by Thermo Fisher Scientific (2 mentions) β mercaptoethanol, by Merck Group (2 mentions) Lsrii flow cytometer, by BD (2 mentions) Anti il 10, by Thermo Fisher Scientific (1 mentions) Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (1 mentions)

12 protocols using anti mhcii fitc

1

Comprehensive Immune Cell Profiling

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Before cytokine staining, T cells underwent in vitro stimulation with PMA (40 ng/ml), Ionomycin (2 μM), Monensin (4 μM) in 1 ml RPMI 1640 (containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G, 0.1 mg/ml streptomycin, and 10 mM HEPES) for 4 h. Cells were resuspended in PBS supplemented with 0.5% BSA and 1 mM EDTA (FACS buffer) (splenocytes and lymphoid cells: 1 × 106/tube, all available CNS mononuclear cells from each sample/tube) and stained with the antibodies listed below at 4˚C for 30 min. The anti-mouse antibodies used for flow cytometric analysis were the following: anti–CD45–APC-Cy7, anti-CD4-APC, anti-IFNγ-BV421, anti-IL-17–PerCP Cy5.5, anti-IL-10–FITC, anti-Foxp3-PE, anti-CD11b-PE-Cy7, anti-Ly6G-BV421, anti-MHC II-FITC, anti-Ly6C-PerCP Cy5.5, anti-CCR2- BV510 (all antibodies were from eBiosciences or BioLegend). After staining, the cells were analyzed with FACSVerse flow cytometer. Flow cytometric data were viewed and analyzed by FlowJo v10.
Hu C.F., Wu S.P., Lin G.J., Shieh C.C., Hsu C.S., Chen J.W., Chen S.H., Hong J.S, & Chen S.J. (2021). Microglial Nox2 Plays a Key Role in the Pathogenesis of Experimental Autoimmune Encephalomyelitis. Frontiers in Immunology, 12, 638381.
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2

Flow Cytometric Analysis of Immune Cells

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The population of each phenotype of immune cells in different groups was evaluated by using flow cytometric analysis as described previously [30 (link)]. In brief, splenocytes were stained with fluorescent antibodies, including anti-CD4-FITC, anti-CD25-PE, anti-Foxp3-PerCP, anti-CD11c-APC, anti-MHCII-FITC, anti-CD86-PE, anti-CD40-PE, anti-CD4-PE (eBiosciences, San Diego, USA), anti-IL-4-APC, anti-CD68-FITC, and anti-CD206-PE (BioLegend, San Diego, USA), according to the manufacturer’s instruction. The FlowJo software was used to analyze the percentages of various phenotypes of immune cells.
Li X., Lan X., Zhao Y., Wang G., Shi G., Li H., Hu Y., Xu X., Zhang B., Ye K., Gu X., Du C, & Wang H. (2019). SDF-1/CXCR4 axis enhances the immunomodulation of human endometrial regenerative cells in alleviating experimental colitis. Stem Cell Research & Therapy, 10, 204.
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3

Multicolor Flow Cytometry of Mouse Immune Cells

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Freshly isolated liver MNCs and spleen cells were initially incubated with anti-mouse CD16/32 (1:100 final dilution; Becton Dickinson) for 15 minutes at room temperature. The washed cells were incubated for 30 minutes at 4 °C with the following fluorescently labeled monoclonal antibodies: anti-ST2L-PE (eBioscience, San Diego, CA), anti-CD11b-APC (eBioscience), anti-CD19-PE-Cy7 (eBioscience), anti-CD4-PerCP (eBioscience), anti-CD3-APC (BD Pharmingen, San Diego, CA), anti-F4/80-FITC (eBioscience), anti-F4/80-PerCP-Cy5.5 (eBioscience), anti-MHCII-FITC (eBioscience). FACS analysis was performed using FACScalibur and/or FACSverse (both from Becton Dickinson).
Peng H., Zhang Q., Li X., Liu Z., Shen J., Sun R., Wei J., Zhao J., Wu X., Feng F., Zhong S., Sun X, & Wu Z. (2016). IL-33 Contributes to Schistosoma japonicum-induced Hepatic Pathology through Induction of M2 Macrophages. Scientific Reports, 6, 29844.
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4

Colon and Caecum Immune Cell Isolation

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Caecum and colon were harvested at autopsy and digested in RPMI containing 5% L-glutamine, 5% penicillin streptomycin, 10% fetal bovine serum (Sigma Aldrich, Dorset, UK), collagenase (1mg/ml), and dispase (0.5mgs/ml, both Gibco, Paisley, UK) for 2 hours at 37 °C. Cells were then forced through a 70μm cell strainer, centrifuged at 405g for 5 minutes and resuspended in 10mls 80% Percoll (GE Healthcare, Buckinghamshire, UK) solution which was then overlaid on a 40% Percoll solution. Cells were centrifuged for 25 minutes at 1000g and the cells at the gradient interface harvested. Fc receptors were blocked using anti-CD16/32 (2 μg/ml E bioscience, Hatfield, UK). Cells were washed and stained with anti-CD45 PeCy7 (1 μg/ml, Becton Dickinson, Oxford, UK), anti-CD103 PE (1 μg/ml), anti-CD11c Alexa700 (2.5 μg/ml), anti-MHC-II FITC (2.5 μg/ml) and anti-F4/80 APC (1 μg/ml) (all E bioscience, Hatfield, UK) and acquired by flow cytometry on the BD LSRII. Data was analysed using FlowJo flow cytometry software (Tree Star inc. Oregon, US).
Bowcutt R., Bramhall M., Logunova L., Wilson J., Booth C., Carding S.R., Grencis R, & Cruickshank S. (2014). A role for the pattern recognition receptor Nod2 in promoting recruitment of CD103+ Dendritic Cells to the colon in response to Trichuris muris infection. Mucosal immunology, 7(5), 1094-1105.
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5

Immune Cell Phenotyping by Flow Cytometry

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The phenotype of various immune cells was evaluated by flow cytometry analysis [FACS, Epic XL, Software Expo32 (Beckman coulter)]. Briefly, splenocytes were stained with fluoresent antibodies, including anti-CD3ε-FITC, anti-CD8a-PerCP, anti-CD4-PE/FITC, anti-CD25-PE, anti-Foxp3-PerCP, anti-CD11c-PE, anti-MHCII-FITC (eBiosciences, San Diego, CA, USA), according to the manufacturer’s instructions. The percentage of each phenotype of immune cells was analyzed with the corresponding Flowjo software.
Lu S., Shi G., Xu X., Wang G., Lan X., Sun P., Li X., Zhang B., Gu X., Ichim T.E, & Wang H. (2016). Human endometrial regenerative cells alleviate carbon tetrachloride-induced acute liver injury in mice. Journal of Translational Medicine, 14, 300.
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6

Dendritic Cell Isolation and Stimulation

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Bone marrow was harvested and cultured as previously described[23 (link)]. On day six of the culture the cells were harvested from the plates and semi-adherent cells removed. The cells were spun at 400g and re-suspended at a concentration of 1×106 cells/ml in DC medium (RPMI 1640 supplemented with 10% LPS-free FBS (Gibco, Paisley, UK) 1% Penicillin/streptomycin and 50mM Beta-mercaptoethanol (Sigma Aldrich, Dorset UK)). The cells were stimulated overnight with LPS (100ng/ml) or T. muris antigen (5μg/ml). After 24 hours the cells were harvested and prepared for flow cytometry. FcR were blocked by incubating cells in anti-CD16/32 (2μg/ml) for 15 minutes. Cells were washed and stained with anti-CD45 PeCy7 (1 μg/ml, Becton Dickinson), anti-CD11c Alexa700 (1μg/ml), anti-MHC-II FITC (2.5 μg/ml) and anti-CD86 PE (1 μg/ml) (all E Bioscience) and acquired using a LSRII flow cytometer (Becton Dickinson Biosciences, Oxfordshire). Data was analysed using Flow Jo flow cytometry analysis software (Tree Star, Inc, Oregon, US).
Bowcutt R., Bramhall M., Logunova L., Wilson J., Booth C., Carding S.R., Grencis R, & Cruickshank S. (2014). A role for the pattern recognition receptor Nod2 in promoting recruitment of CD103+ Dendritic Cells to the colon in response to Trichuris muris infection. Mucosal immunology, 7(5), 1094-1105.
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7

Colon and Caecum Immune Cell Isolation

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Caecum and colon were harvested at autopsy and digested in RPMI containing 5% L-glutamine, 5% penicillin streptomycin, 10% fetal bovine serum (Sigma Aldrich, Dorset, UK), collagenase (1mg/ml), and dispase (0.5mgs/ml, both Gibco, Paisley, UK) for 2 hours at 37 °C. Cells were then forced through a 70μm cell strainer, centrifuged at 405g for 5 minutes and resuspended in 10mls 80% Percoll (GE Healthcare, Buckinghamshire, UK) solution which was then overlaid on a 40% Percoll solution. Cells were centrifuged for 25 minutes at 1000g and the cells at the gradient interface harvested. Fc receptors were blocked using anti-CD16/32 (2 μg/ml E bioscience, Hatfield, UK). Cells were washed and stained with anti-CD45 PeCy7 (1 μg/ml, Becton Dickinson, Oxford, UK), anti-CD103 PE (1 μg/ml), anti-CD11c Alexa700 (2.5 μg/ml), anti-MHC-II FITC (2.5 μg/ml) and anti-F4/80 APC (1 μg/ml) (all E bioscience, Hatfield, UK) and acquired by flow cytometry on the BD LSRII. Data was analysed using FlowJo flow cytometry software (Tree Star inc. Oregon, US).
Bowcutt R., Bramhall M., Logunova L., Wilson J., Booth C., Carding S.R., Grencis R, & Cruickshank S. (2014). A role for the pattern recognition receptor Nod2 in promoting recruitment of CD103+ Dendritic Cells to the colon in response to Trichuris muris infection. Mucosal immunology, 7(5), 1094-1105.
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8

Comprehensive Antibody Validation for Research

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The following antibodies were used for Western blot analysis: anti‐SMAD4 (CST, 46535), anti‐STING (CST, 13647), anti‐RAD51 (CST, 8875), anti‐β2‐microglobulin (Abcam, ab75853), anti‐IRF3 (CST, 4302), anti‐phosphorylated‐IRF3 (CST, 29047), anti‐TBK1 (CST, 13504), anti‐phosphorylated‐TBK1 (CST, 5483), anti‐γ‐H2AX (CST, 9718), and anti‐β‐ACTIN (Sigma, A3854). Anti‐MHC‐I‐Eflour 450 (eBioscience, 48‐5999‐82) and anti‐MHC‐II‐FITC (eBioscience, 11‐5321‐82) were purchased from Thermo Fisher Scientific (USA). The neutralizing antibodies used for in vivo experiment were: Anti‐CD8 (BE0004‐1), anti‐CD4 (BE0003‐1), anti‐IFNAR1(BE0241), anti‐NK1.1(BE0036) and Rat lgG2a isotype control (BE0089) antibody, all purchased from BioXCell Inc. DMSO was obtained from Sigma‐Aldrich (St. Louis, MO), Murine IFN‐β was from R&D, Inc. (8234‐MB‐010), and murine TGF‐β1 was from Peptrotech Inc. (100‐21‐50).
Xiong W., He W., Wang T., He S., Xu F., Wang Z., Wang X., Guo H., Ling J., Zhang H., Liu Y., Xing K., Li M., Zhang H., Li J., Niu N., Xue J., Zhan Q., Liu Z., Bei J., Huang P., Liu J., Xia L, & Xia X. (2022). Smad4 Deficiency Promotes Pancreatic Cancer Immunogenicity by Activating the Cancer‐Autonomous DNA‐Sensing Signaling Axis. Advanced Science, 9(7), 2103029.
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9

Bone Marrow-Derived Dendritic Cell Phenotyping

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Day 12 bone marrow DC (BMDC) were collected and labeled for 30 min in PBS + 1% BSA (Bovine Serum Albumin, cat. K45-001, GE Healthcare Bio-Sciences, Pasching, Austria) containing anti-CD11c-APC (eBioscience, clone N418, 1:400), anti-MHCII-FITC (eBioscience, clone M5/114.15.2, 1:1500), and anti-CD40-PE (BD Biosciences, clone 3/23, 1:200), or anti-CD86-PE (eBioscience, clone GL1, 1:200) or anti-CD274-PE (PD-L1, eBioscience, clone M1H5, 1:400). As control, corresponding isotype control antibodies (eBioscience, San Diego, CA) were used. Surface labeling was analyzed using a FACS Calibur Flow Cytometer (BD Biosciences, San Jose, CA). Data analysis was performed using FCS Express V3 (DeNovo Software, Glendale, CA).
Driedonks T.A., van der Grein S.G., Ariyurek Y., Buermans H.P., Jekel H., Chow F.W., Wauben M.H., Buck A.H., ‘t Hoen P.A, & Nolte-‘t Hoen E.N. (2018). Immune stimuli shape the small non-coding transcriptome of extracellular vesicles released by dendritic cells. Cellular and Molecular Life Sciences, 75(20), 3857-3875.
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10

Quantification of Regulatory T Cells

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For detection of CD4+Foxp3+ Tregs, spleens, blood, and kidneys of C57BL/6 mice were subjected to flow cytometry analysis as described previously [14 (link)]. Anti-CD4- fluorescein isothiocyanate (FITC), anti-CD25-Phycoerythrin (PE), anti-Foxp3-efluor450, anti-IL-17A-allophycocyanin (APC), anti-CD11c-PE, anti-MHCII-FITC antibodies were purchased from eBioscience (USA). FACS Attune Nxt (Life, Thermo) was used for the analysis.
Liang Y., Xue N., Wang X., Ding X, & Fang Y. (2019). Superagonistic CD28 protects against renal ischemia injury induced fibrosis through a regulatory T-cell expansion dependent mechanism. BMC Nephrology, 20, 407.
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