Apc cy7 anti cd45 30 f11

Manufactured by BioLegend

APC-Cy7-anti-CD45 (30-F11) is a fluorescently labeled antibody that targets the CD45 antigen. CD45 is a transmembrane protein tyrosine phosphatase expressed on the surface of most hematopoietic cells.

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Anti-CD45 APC/Cy7 (clone 30-F11; CD45-APC/Cy7 (30-F11, Anti-CD45 (30-F11, CD45 (30-F11, Anti-CD45-APC-Cy7 CD45-APC-Cy7 Anti-CD45-APC CD45-APC APC-Cy7 Anti-CD45

5 protocols using apc cy7 anti cd45 30 f11

Kupffer Cell and MDSC Characterization

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Cells were incubated with Fc-blocking reagent (TrueStain, Biolegend) for 5 min, and subsequently stained for 20 min at 4°C with the relevant antibody. Some antibodies were used to analyze Kupffer cells: FITC anti-F4-80 (BM8), APC anti-I-A-I-E (M5/114.15.2), PE anti-CD86 (GL-1), PECy7 anti-CD68 (FA-11), PerCP anti-CD11b (M1/70) (Biolegend), PE anti-CD273 (122), and anti-CD274 (MIH5) (eBiosciences). For experiments involving Kupffer cell/MDSC co-culture, Kupffer cells were first labeled with CFSE (0.5 µM, Invitrogen). MDSC were characterized with the following antibodies: APC anti-CCR2 (475301, R&D Systems), APC anti-CD244.2 (eBio244F4, eBiosciences), APC anti-CD54 (YN1/1.7.4), APC anti-CD115 (AFS98), PE anti-Ly6C (HK1,4), PerCP anti-CD11b (M1/70), AlexaFluor488 anti-CD146 (ME-9F1), PECy7 anti-Gr1 (RB6-8C5), APC Cy7 anti-CD45 (30-F11, Biolegend). All samples were acquired with a Flow Cytometer Cyan (Beckman Coulter), and analyzed with FloJo (Treestar).

Lacotte S., Slits F., Orci L.A., Meyer J., Oldani G., Delaune V., Gonelle-Gispert C., Morel P, & Toso C. (2016). Impact of myeloid-derived suppressor cell on Kupffer cells from mouse livers with hepatocellular carcinoma. Oncoimmunology, 5(11), e1234565.

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Isolation and Analysis of Leukocytes

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The lung and the brain were harvested after animal euthanasia, washed in PBS, minced into small pieces and treated with 0.5 mg/ml Collagenase IV (Sigma-Aldrich, St. Louis, MO, USA) and 30 μg/ml DNase (Sigma-Aldrich) in RPMI 1640 for 30 min at 37°C. The tissues were then forced through a 100 μm cell strainer and layered onto 30% Percoll (GE, Pittsburg, PA, USA) for centrifugation at 1500 g, 30 min, at room temperature. Leukocyte pellets were treated with ACK (Ammonium-Chloride-Potassium) lysing buffer (BioLegend) for 3 min to lyse red blood cells, washed with PBS, and placed on ice until use. For flow cytometry staining, Fc receptors were blocked using anti-CD16/32 Abs (93; eBioscience, San Diego, CA, USA). The cells were stained using APC-Cy7-anti-CD45 (30-F11, BioLegend) and APC-anti-Ly6G (1A8, eBioscience). In some experiments, the cells were also stained using Acti-stain 555 phalloidin (to label F-actin, Cytoskeleton, Denver, CO, USA). Flow cytometry was performed using a FACSAria II flow cytometer (BD Biosciences, San Jose, CA, USA); the data obtained were subsequently analyzed using FlowJo (Tree Star, Ashland, OR, USA).

Sun D., Zhang M., Liu G., Wu H., Li C., Zhou H., Zhang X, & Shi M. (2016). Intravascular clearance of disseminating Cryptococcus neoformans in the brain can be improved by enhancing the recruitment of neutrophils. European journal of immunology, 46(7), 1704-1714.

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Murine Leukocyte Purification and Characterization

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Leukocytes were purified from the lung or bone marrow of mice as previously described [17 (link)]. For flow cytometry staining, Fc receptors were blocked using anti-CD16/32 Abs (93; eBioscience). The cells were stained using APC-Cy7-anti-CD45 (30-F11, BioLegend), FITC-anti-Ly6G (1A8, BioLegend), PE-Cy7-anti-CD11b (M1/70, BioLegend), and Brilliant Violet 421-anti-CD11c (N418, BioLegend). Neutrophils were defined as CD45⁺ Ly6G⁺CD11b⁺CD11c⁻.

Sun D, & Shi M. (2016). Neutrophil swarming toward Cryptococcus neoformans is mediated by complement and leukotriene B4. Biochemical and biophysical research communications, 477(4), 945-951.

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Flow Cytometric Analyses of Immune Cells

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Antibodies for flow cytometric analyses were as follows.
For staining mouse cells, anti-FcεRIα-fluorescent isothiocyanate (FITC; clone, Mar-1), anti-CD64 (FcγRI)-Phycoerythrin (PE; X54-5/7.1), anti-F4/80-PE (BM8), anti-CD16/32 (FcγRIII/II)-PE/Cyanine 7 (Cy7; 93), anti-Gr-1-Allophycocyanin (APC; RB6-8C5), and anti-CD45- APC/Cy7 (30-F11) were from Biolegend (San Diego, CA). For human NK cell staining, anti-CD45-Brilliant Violet (BV) 510 (HI30), anti-CD3-BV421 (UCHT1), anti-CD56-AlexaFluor700 (HCD56), anti-CD56-PE/Cy7 (HCD56), anti-CD16-BV605 (3G8), and anti-CD335 (NKp46)-PE (9E2) were from Biolegend. Anti-CD19-BUV395 (SJ25C1), anti-CD14-APC/Cy7 (HCD14), and anti-CD314 (NKG2D)-APC (1D11) were from BD Biosciences (San Jose, CA). For detecting human IgG captured by mouse cells, anti-human IgG-FITC (G18-145, BD Biosciences) was used.
Anti-HER2 antibody (Trastuzumab, Herceptin®) and anti-CD20 antibody (Rituximab, Rituxan®) were purchased from Chugai (Tokyo, Japan). Anti-CCR4 antibody (Mogamulizumab, Poteligeo®) was from Kyowa-Kirin (Tokyo, Japan).

Katano I., Ito R., Kawai K, & Takahashi T. (2020). Improved Detection of in vivo Human NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity Using a Novel NOG-FcγR-Deficient Human IL-15 Transgenic Mouse. Frontiers in Immunology, 11, 532684.

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Multiparameter Flow Cytometry Analysis

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For FACS analysis, the following antibodies were used: anti-CD3-Bv421-(clone 17A2, Biolegend), anti-CD4-Bv421 (clone RM 4-5, eBioscience), anti-CD8-FITC (clone 53-6.7, Biolegend), anti-CD25 PE (clone PC61, Biolegend), anti-CD27-APC (clone LG.3A10, Biolegend), anti-CD11b-FITC (clone M1/70, Biolegend), anti-CD11b-Bv421 (clone M1/70, Biolegend), anti-FceRIα-PE (MAR-1, Biolegend), anti-F4/80-PE (BM8, Biolegend), anti-CD45-APC/Cy7 (30-F11, Biolegend), anti-CD107a-FITC (1D4B, Biolegend) and anti-P2X7-AF647 (clone Hano44, UKE) (Adriouch et al., 2005) (link). Flow cytometric analyses were performed on a BD Fortessa (Beckton Dickinson) or a BD FACS CantoII (Beckton Dickinson).

Er-Lukowiak M., Duan Y., Rassendren F., Ulmann L., Nicke A., Ufer F., Friese M.A., Koch‐Nolte F., Magnus T., & Rissiek B. (2020). A P2rx7 passenger mutation affects the vitality and function of immune cells in P2X4ko and other transgenic mice.

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