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Anti mouse cd45 30 f11

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Anti-mouse CD45 (30-F11) is an antibody that recognizes the mouse CD45 antigen. CD45 is a tyrosine phosphatase that is expressed on the surface of most hematopoietic cells, including T cells, B cells, and myeloid cells. This antibody can be used to identify and distinguish different cell populations in flow cytometry applications.

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Lab products found in correlation

Cd11b m1 70, by BioLegend (2 mentions) Fixable viability dye efluor 455, by Thermo Fisher Scientific (1 mentions) Ly6g 1a8, by BioLegend (1 mentions) Ly6c hk1, by BioLegend (1 mentions) Collagenase d, by Roche (1 mentions) Fortessa, by BD (1 mentions) Deoxyribonuclease 1, by Roche (1 mentions) Foxp3 staining kit, by BioLegend (1 mentions) Cd3 17a2, by BioLegend (1 mentions) Cd64 x54 5 7, by BioLegend (1 mentions) F4 80 bm8, by BioLegend (1 mentions) Mhcii m5 114.15.2, by Thermo Fisher Scientific (1 mentions) Ly6c al 21, by BD (1 mentions) B220 ra3 6b2, by BioLegend (1 mentions) Lineage cocktail, by BD (1 mentions) Facsaria iiu, by BD (1 mentions) Facscalibur, by BD (1 mentions) Cell culture media and reagents, by GE Healthcare (1 mentions) Bio gel polyacrylamide beads p 100 fine 45 90 μm, by Bio-Rad (1 mentions) Ionomycin, by Fujifilm (1 mentions)

9 protocols using anti mouse cd45 30 f11

1

Characterizing Immune Cells in Arthritic Mice

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CAIA mice were intravenously injected with control IgG, unmodified α-TNF, or CBP–α-TNF at a dose of 200 μg per mouse. On day 5, hind paws were harvested and digested in Dulbecco’s modified eagle medium supplemented with 2% FBS, collagenase D (2 mg/ml) and deoxyribonuclease I (40 μg/ml; Roche) for 30 min at 37°C. Single-cell suspensions were obtained by gently disrupting through a 70-μm cell strainer. Antibodies against the following molecules were used: anti-mouse CD45 (30-F11, BD Biosciences), F4/80 (T45-2342, BD Biosciences), Ly6G (1A8, BioLegend), Ly6C (HK1.4, BioLegend), and CD11b (M1/70, BioLegend). Fixable live/dead cell discrimination was performed using Fixable Viability Dye eFluor 455 (eBioscience) according to the manufacturer’s instructions. Following a washing step, cells were stained with specific antibodies for 20 min on ice. Flow cytometric analyses were done using a Fortessa (BD Biosciences) flow cytometer and analyzed using FlowJo software (Tree Star).
Katsumata K., Ishihara J., Mansurov A., Ishihara A., Raczy M.M., Yuba E, & Hubbell J.A. (2019). Targeting inflammatory sites through collagen affinity enhances the therapeutic efficacy of anti-inflammatory antibodies. Science Advances, 5(11), eaay1971.
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2

Comprehensive Immunophenotyping by Flow Cytometry

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Flow cytometry was performed with LSR II (BD Biosciences) using various combinations of the following mAbs: anti-human CD45 (2D1), CD19 (HIB 19), CD3 (UCHT1), CD4 (RPA-T4), CD8 (SK1), CD33 (WM53), CCR7 (G043H7), CD45RA (HI100), CD31 (WM59), CD127 (A019D5), CD25 (M-A251), CD235a (HI264); anti-mouse CD45 (30-F11), and TER119 (TER-119); and isotype control mAbs (purchased from BD Biosciences PharMingen or Biolegend). Intracellular FoxP3 staining was performed with FoxP3 Staining Kit (Biolegend) according to the manufacturer's instructions.
Yoshihara S., Li Y., Xia J., Danzl N., Sykes M, & Yang Y.G. (2019). Posttransplant Hemophagocytic Lymphohistiocytosis Driven by Myeloid Cytokines and Vicious Cycles of T-Cell and Macrophage Activation in Humanized Mice. Frontiers in Immunology, 10, 186.
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3

Comprehensive Immune Cell Profiling

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In addition to critical reagents listed throughout this section, the following antibodies were used for flow cytometry and confocal imaging: anti–mouse CD45 (30-F11; BD Biosciences), CD3 (17A2; BioLegend), TCR Vγ5 (536; BioLegend), MHCII (M5/114.15.2; eBioscience), F4/80 (BM8; BioLegend), CD64 (X54-5/7.1; BioLegend), CD11b (M1/70; BioLegend), and Ly6C (AL-21; BD Biosciences).
Gentek R., Ghigo C., Hoeffel G., Jorquera A., Msallam R., Wienert S., Klauschen F., Ginhoux F, & Bajénoff M. (2018). Epidermal γδ T cells originate from yolk sac hematopoiesis and clonally self-renew in the adult. The Journal of Experimental Medicine, 215(12), 2994-3005.
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4

Murine Bone Marrow Cell Phenotyping

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Murine long bone marrows were flushed with flow cytometry staining buffer (FSB) (2% FBS, 2 mM EDTA in 1× PBS) immediately after sacrifice and 1 × 106 cells were incubated with fluorescent-conjugated antibodies (PE 5 μl; FITC 2 μl; APC 4 μl) in 100 μl of FACS buffer for 20–30 min at 4 °C. Intracellular staining of CD68 utilized antibody specific reagents for a 15-min fixation followed by a 30 min incubation with the CD68 antibody in permeabilization buffer. Cells from all stains were then washed, and analyzed on a FACS Calibur or a FACS Aria IIu (BD Biosciences, San Jose, CA).
Antibodies for flow cytometric analyses included the following: Antimouse CD45 (30-F11), Lineage cocktail, sca1 (E13-161.7), CD29 (HM β1-1), and the Annexin PI kit were obtained from BD Biosciences (San Jose, CA). Antimouse CD11b (M1/70, APC) was obtained from eBioscience (San Diego, CA). Antimouse CD68 (FA-11) antibody and respective fixation and permeabilization reagents were purchased from Abd Serotec (Alexa Fluor 647; Leucoperm Reagents; Raleigh, NC) or Biolegend (FITC; Fixation Buffer/Permeabilization Buffer; San Diego, CA). B220 (RA3-6B2) was obtained from Biolegend.
Koh A.J., Sinder B.P., Entezami P., Nilsson L, & McCauley L.K. (2017). The skeletal impact of the chemotherapeutic agent etoposide. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA, 28(8), 2321-2333.
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5

Myeloid Cell Isolation and Characterization

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Bio-gel polyacrylamide beads (P-100 fine, 45–90 μm) were purchased from Bio-Rad (Hercules, CA, USA); anti-mouse CD45.1 (A20), CD45.2 (104), CD11b (M1/70), CD115 (AFS98), lymphocyte antigen 6 complex, locus C (Ly-6C) (HK1.4), and lymphocyte antigen 6 complex, locus G (Ly-6G) (1A8) were purchased from BioLegend (San Diego, CA, USA); anti-mouse CD45 (30-F11) was obtained from BD Biosciences (San Jose, CA, USA); quantitative PCR (qPCR) primers were purchased from Qiagen (Germantown, MD, USA); and all cell culture media and reagents were obtained from GE Healthcare (Waukesha, WI, USA).
Kapellos T.S., Taylor L., Feuerborn A., Valaris S., Hussain M.T., Rainger G.E., Greaves D.R, & Iqbal A.J. (2019). Cannabinoid receptor 2 deficiency exacerbates inflammation and neutrophil recruitment. The FASEB Journal, 33(5), 6154-6167.
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6

Multiparameter Flow Cytometry Analysis

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Anti-mouse CD45 (30-F11) and IL-17A (TC11-18H10) antibodies were obtained from BD Biosciences (Franklin Lakes, NJ). Anti-mouse CD11b (M1/70), CD11c (N418), TCRγδ (eBioGL3), and Ki67 (SolA15) antibodies were purchased from eBioscience (San Diego, CA). Anti-mouse F4/80 (BM8), Ly-6G (1A8), MHC class II (M5/114.15.2), TCRβ (H57-597), and Vγ4 (UC3-10A6) antibodies were purchased from BioLegend (San Diego, CA). For intracellular staining, cells were stimulated for 3 h with 50 ng/ml PMA (phorbol myristate acetate; Sigma-Aldrich, St Louis, MO) and 1 μg/ml ionomycin (Wako, Osaka, Japan) in the presence of 10 μg/ml brefeldin A (Sigma-Aldrich) or put 10 μg/ml brefeldin A in the collagenase solution, and then fixed and permeabilized with Cytofix/Cytoperm buffer (BD Biosciences). Flow cytometry was performed using LSRFortessa (BD Biosciences) and analyzed with FlowJo (TreeStar, San Carlos, CA).
Nakamizo S., Honda T., Adachi A., Nagatake T., Kunisawa J., Kitoh A., Otsuka A., Dainichi T., Nomura T., Ginhoux F., Ikuta K., Egawa G, & Kabashima K. (2017). High fat diet exacerbates murine psoriatic dermatitis by increasing the number of IL-17-producing γδ T cells. Scientific Reports, 7, 14076.
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7

Multiparametric Flow Cytometry Panel

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The following antibodies from BioLegend were used for flow cytometry: anti-human CD2 (TS1/8), CD3 (HIT3a), CD10 (HI10a), CD33 (WM53), CD34 (581), CD36 (5–271), CD38 (HB-7), CD45 (H130), CD45RA (HI100), CD56 (HCD56), CD94 (DX22), CD122 (TU27), CD132 (TUGh4), CD135 (BV10A4H2), NKG2D (1D11) and NKp46 (9E2). Anti-human NKG2A antibody was from R&D systems (Minneapolis, MN). Anti-mouse CD45 (30-F11) was acquired from BD Biosciences. For flow cytometry, samples were stained with antibodies in 50 μl flow buffer (0.2% bovine serum albumin [BSA, sigma], 0.05% sodium azide [sigma] in PBS) on ice for 30 mins. Stained samples were then analyzed on a BD LSR II flow cytometer, and data analyzed with FACS Diva (BD Biosciences) and FlowJo (TreeStar version e.g. 7.6.5). Isotype-matched control antibodies were used for all fluorochrome-isotype combinations. For fluorescent-activated cell sorting (FACS), cells were stained with appropriate antibodies in RoboSep buffer (Stemcell Technologies), and sorted on a BD FACSAria II (BD Bioscience).
Chen Q., Ye W., Jian Tan W., Mei Yong K.S., Liu M., Qi Tan S., Loh E., TE Chang K., Chye Tan T., Preiser P.R, & Chen J. (2015). Delineation of Natural Killer Cell Differentiation from Myeloid Progenitors in Human. Scientific Reports, 5, 15118.
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8

Isolation and Characterization of Epidermal Cells

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To generate single cell suspensions, epidermal scrapings were resuspended in complete RPMI, vortexed, pipetted to disaggregate cells, and filtered (40 μm strainer). For antigen re-expression, cells were rested overnight at 37° C with 5% CO2 as described (15 (link)). Before staining, cells were filtered as above and counted with a Vi-cell XR (Beckman Coulter). We used validated commercial reagents: LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies), anti-mouse CD3 (17A2), CD25 (PC61.5), granzyme B (GzA-3G8.5), Lamp-1 (eBio1D4B), NK1.1 (PK136), IL-17A (XMG1.2), IL-22 (1H8PWSR) (Ebioscience), anti-mouse γδ TCR (GL3), CXCR3 (CXCR3-173), CD69 (H1.2F3), Vγ5 (536), Vγ4 (UC3-10A6), Vγ1 (2.11) (Biolegend), anti-mouse CD45 (30-F11) (BD Biosciences), on Becton-Dickinson LSR II hardware using FACSDiva software. For cytokine staining, cells were stimulated with Leukocyte Activation Cocktail with Golgiplug (BD Biosciences) for 5 h, surface stained, fixed and permeabilized with Foxp3/transcription factor buffer (Ebioscience), then stained.
Dao V., Liu Y., Pandeswara S., Svatek R.S., Gelfond J.A., Liu A., Hurez V, & Curiel T.J. (2016). Immune stimulatory effects of rapamycin are mediated by stimulation of antitumor γδ T cells. Cancer research, 76(20), 5970-5982.
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9

Isolation and Activation of DC Subsets

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DC subsets for vaccination were isolated from spleens and bone marrow of humanized non-tumor-bearing littermates treated with Flt3L as previously described.22 (link) Briefly, DCs were enriched by negative selection from bone marrow cells by incubation with anti-mouse Ter119 (TER-119), anti-human CD14 (RMO52, Beckman Coulter), anti-human CD19 (J3-119, Beckman Coulter), anti-human CD3 (OKT3, BioXCell), anti-human CD34 (My10, BD), and anti-mouse CD45 (30-F11, BD) followed by removal of antibody-bearing cells using sheep anti-rat IgG Dynabeads (Invitrogen). Enriched cells were stained with anti-human CD45-APC-Cy7, anti-HLA-DR-PECy7 (L243), anti-CD123-PerCP-Cy5.5 (6H6), anti-CD141-APC (M80), anti-CD1c-PE (L161), anti-CD11c-PE-CF594, and anti-CD3/CD19/CD20-Pacific Blue. DC subsets were sorted as CD3/CD14/CD19/CD20–HLA-DR+CD11c+CD141+ (cDC1) and CD3/CD14/CD19/CD20–HLA-DR+CD11c+CD141– (cDC2) using a MoFlo Astrios cell sorter (Beckman Coulter). DCs were activated for 2 hours with 10 µg/mL PolyIC prior to intratumoral injection of 20-25 x 103 DCs per mouse.
Lee Y.S., O'Brien L.J., Walpole C.M., Pearson F.E., Leal-Rojas I.M., Masterman K.A., Atkinson V., Barbour A, & Radford K.J. (2021). Human CD141+ dendritic cells (cDC1) are impaired in patients with advanced melanoma but can be targeted to enhance anti-PD-1 in a humanized mouse model. Journal for Immunotherapy of Cancer, 9(3), e001963.
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