Anti mhc 2 fitc

Manufactured by Miltenyi Biotec

Anti-MHC II-FITC is a fluorescently-labeled antibody that binds to major histocompatibility complex (MHC) class II molecules. It is used for the detection and analysis of MHC class II-expressing cells in flow cytometry applications.

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3 protocols using anti mhc 2 fitc

Multiparametric Flow Cytometry of Immune Cells

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Cervical lymph node cells were isolated, washed, counted, and resuspended at 100,000 cells in 50 μL of flow cytometry buffer. Cells were treated with FcR-block (Miltenyi Biotec, Bergisch Gladbach, Germany) and labeled for surface markers via antibodies, per the manufacturer’s instructions^{31 (link),39 (link)}. Dead cells were excluded from analysis by labeling with propidium iodide viability dye (Miltenyi Biotec), per the manufacturer’s instructions^{31 (link),39 (link)}. Neutrophils: anti-CD11b-APC (Miltenyi Biotec; clone REA592), anti-Ly6C-FITC (Miltenyi Biotec; clone REA796), anti-Ly6G-VioBlue (Miltenyi Biotec; clone 1A8). M1-macrophages: anti-CD11b-APC (Miltenyi Biotec; clone REA592), anti-MHC II-FITC (Miltenyi Biotec; clone REA528), anti-CD206-PE (Miltenyi Biotec; clone MR6F3), anti-CD64-APC-Vio770 (Miltenyi Biotec; clone REA286). Plasmacytoid dendritic cells: anti-CD11c-PE-Vio770 (Miltenyi Biotec; clone REA754), anti-MHC II-FITC (Miltenyi Biotec; clone REA528), anti-B220-VioBlue (Miltenyi Biotec; clone REA755). A minimum of 5,000 gated cells were analyzed per specimen. Data was acquired by the MACSQuant System (Miltenyi Biotec) and analyses were performed via FlowJo 11.0 software (TreeStar, Ashland, OR, USA), as previously reported^{31 (link),39 (link)}.

Swanson B.A., Carson M.D., Hathaway-Schrader J.D., Warner A.J., Kirkpatrick J.E., Corker A., Alekseyenko A.V., Westwater C., Aguirre J.I, & Novince C.M. (2021). Antimicrobial-Induced Oral Dysbiosis Exacerbates Naturally Occurring Alveolar Bone Loss. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 35(11), e22015.

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Retinal Immune Cell Isolation and Characterization

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Retinas were dissected out and homogenized in 500 μL of PBS (phosphate-buffered saline) with liberase TL at 0.8 wunsch/mL (Sigma-Aldrich) for 30 min at 37 °C and 5% CO₂. The retinal homogenate was washed with PBS and the pellet containing the immune cells was re-suspended in 100 μL PBS containing 1 μL of Viobility 405/520 Fixable Dye (Miltenyi). Cells were washed and labeled with 50 μL of primary antibodies mix: anti-CD45-VioBlue (REA737), anti-MHCII-FITC (REA813), anti-CD11b-PE (REA592), anti-Ly-6C-PE-Vio770 (REA796), anti-CD3-APC (REA641), and anti-Ly-6g-APC-Vio770 (REA526) (Miltenyi). After labeling, cells were fixed in 4% paraformaldehyde. For compensation settings, single-stained cellular controls with corresponding antibodies were used. Fluorescence intensities were measured using a MACSQuant analyzer (Miltenyi) and data were analyzed using the FlowJo Software.

Conart J.B., Blot G., Augustin S., Millet-Puel G., Roubeix C., Beguier F., Charles-Messance H., Touhami S., Sahel J.A., Berrod J.P., Léveillard T., Guillonneau X., Delarasse C, & Sennlaub F. (2020). Insulin inhibits inflammation-induced cone death in retinal detachment. Journal of Neuroinflammation, 17, 358.

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Immune Cell Subset Analysis Protocol

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Cells were treated with FcR‐block (Miltenyi Biotec) and stained for cell surface markers. Myeloid‐derived suppressor cells (MDSCs): anti‐CD11b‐APC (Miltenyi Biotec; clone REA592), anti‐Ly6G‐VioBlue (Miltenyi Biotec; clone 1A8), anti‐F4/80‐PE (Miltenyi Biotec; clone REA126), anti‐Ly6C‐FITC (Miltenyi Biotec; clone REA796). Plasmacytoid dendritic cells (pDC): anti‐CD11c‐PE‐Vio770 (Miltenyi Biotec; clone REA754), anti‐MHC II‐FITC (Miltenyi Biotec; clone REA528), anti‐B220‐VioBlue (Miltenyi Biotec; clone REA755). M1/M2 macrophages: anti‐CD11b‐APC (Miltenyi Biotec; clone REA592), anti‐CD11c‐PE‐Vio770 (Miltenyi Biotec; clone REA754), anti‐MHC class II‐FITC (Miltenyi Biotec; clone REA528), anti‐CD64‐APC‐Vio770 (Miltenyi Biotec; clone REA286), anti‐B220‐VioBlue (Miltenyi Biotec; clone REA755), anti‐CD206‐PE (eBioscience, Santa Clara, CA, USA; clone MR6F3). Dead cells were excluded from analysis via propidium iodide viability dye (Miltenyi Biotec).

Hathaway‐Schrader J.D., Poulides N.A., Carson M.D., Kirkpatrick J.E., Warner A.J., Swanson B.A., Taylor E.V., Chew M.E., Reddy S.V., Liu B., Westwater C, & Novince C.M. (2020). Specific Commensal Bacterium Critically Regulates Gut Microbiota Osteoimmunomodulatory Actions During Normal Postpubertal Skeletal Growth and Maturation. JBMR Plus, 4(3), e10338.

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