Cd45 clone 30 f11

Manufactured by Thermo Fisher Scientific

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CD45 (clone 30-F11) is a laboratory reagent used for the identification and enumeration of leukocytes (white blood cells) in biological samples. It is a monoclonal antibody that binds specifically to the CD45 antigen, a transmembrane protein expressed on the surface of all human leukocytes. This reagent can be used in flow cytometry applications to distinguish and quantify different types of white blood cells.

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CD45 (30-F11, (34 citations) Clone 30-F11 (22 citations) CD45-eFluor450 (clone 30-F11, (4 citations) APC-Cy7-anti-CD45 (clone 30-F11), (3 citations) CD45 PerCP/CY5.5 clone 30-F11 (3 citations) Anti-mouse CD45 (clone 30-F11, (2 citations) CD45-AF700 (clone 30-F11; (2 citations) CD45-PE (clone 30-F11) (2 citations) CD45-biotin (clone 30-F11, (2 citations) EFluor 450 anti-CD45 (clone 30-F11), (2 citations)

31 protocols using cd45 clone 30 f11

Characterization of Tumor-Infiltrating Lymphocytes

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Single cell suspensions from spleens and lymph nodes were prepared as described.6 (link) TIL were isolated from pooled tumors as described.26 (link) All flow cytometry experiments were performed at least 3 times. Single cell suspensions were incubated with mouse Fc receptor binding inhibitor for 10 minutes before staining with antibodies to CD45 (clone 30-F11), CD3 (clone 145-2C11), CD4 (clone GK1.5), CD8 (clone 53-6.7), CD19 (clone eBio1D3), CD86 (clone GL1), CD11b (clone M1/70), Gr-1 (clone RB6-8C5), CD69 (clone H1.2F3), CD44 (clone IM9), CD62L (clone MEL-14), and CD11c (clone N418; all from eBioscience, San Diego, CA) for 30 minutes. Flow cytometry was performed using FACS Calibur (BD Biosciences) and the lymphocyte population was selected by gating CD45⁺ cells. The data were analyzed using Flow Jo software (Tree Star, Ashland, OR). For tetramer staining, 10 μL of PE labeled HLA-A*02:01 Human HPV16 E7 tetramer (NIH Tetramer Core Facility) was added to 200 μL mouse lymphocyte suspension (1×106 cells per tube). After incubation for 30 minutes, cells were centrifuged and resuspended in phosphate-buffered saline with 1% paraformaldehyde and then analyzed by flow cytometry. PE labeled HLA-A*02:01 human mesothelin tetramer (NIH Tetramer Core Facility) was used as control.

Dai M., Hellstrom I., Yip Y.Y., Sjögren H.O, & Hellstrom K.E. (2018). Tumor Regression and Cure Depends on Sustained Th1 Responses. Journal of Immunotherapy (Hagerstown, Md. : 1997), 41(8), 369-378.

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Isolation and Analysis of Midbrain Mononuclear Cells

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Mononuclear cells were isolated 4 weeks post-transduction from ventral midbrains with bilateral AAV injections, according to published protocols [27 (link), 30 (link)]. Briefly, midbrains were digested with 1 mg/mL Collagenase IV (Sigma) and 20 μg/mL DNAse I (Sigma) diluted in RPMI 1640 with 10% heat inactivated fetal bovine serum, 1% L-glutamine (Sigma), and 1% Penicillin-Streptomycin (Sigma). Mononuclear cells were separated out using a 30/70% Percoll gradient, as previously described [30 (link)]. Isolated cells were blocked with anti-Fcy receptor (clone 2.4G2 BD Biosciences) then incubated with fluorescent-conjugated antibodies against CD45 (clone 30-F11, eBioscience), CD11b (clone M1/70, BioLegend), MHCII (M5/114.15.2, BioLegend), Ly6C (clone HK 1.4, BioLegend), CD4 (clone GK1.5, BioLegend), and CD8a (clone 53-6.7, BioLegend). A fixable viability dye was used to distinguish live cells from debris per manufacturer’s instructions (Fixable Near-IR LIVE/DEAD Stain Kit, Invitrogen). Samples were analyzed using an Attune Nxt flow cytometer (Thermo Fisher Scientific) and FlowJo software (Tree Star).

Williams G.P., Schonhoff A.M., Jurkuvenaite A., Thome A.D., Standaert D.G, & Harms A.S. (2018). Targeting of the class II transactivator attenuates inflammation and neurodegeneration in an alpha-synuclein model of Parkinson’s disease. Journal of Neuroinflammation, 15, 244.

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Multiparametric Flow Cytometry Immunophenotyping

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Aliquots of 1 × 10⁶ cells were stained with different monoclonal antibodies according to standard protocols. The cells were analyzed on a FACSVerse cytometer (BD Biosciences, San Diego, CA, USA). Fluorescent antibodies of CD45 (clone 30-F-11), CD3ε (clone 145-2C11), CD4 (clone GK1.5), CD83 (clone Michel-19), CD11b (clone M1/70), ly6c(clone HK1.4), F4/80 (clone BM8), B220 (clone RA3-6B2), NK1.1 (clone PK136), MHC-II (clone M5/114.15.2), CD11c (clone N418), CD69 (clone H1.2F3), and Ki67 (clone B56) conjugated with the corresponding fluorescent dyes (eBioscience, San Diego, CA, USA) were used in the experiments.

Lin W., Zhou S., Feng M., Yu Y., Su Q, & Li X. (2021). Soluble CD83 Regulates Dendritic Cell–T Cell Immunological Synapse Formation by Disrupting Rab1a-Mediated F-Actin Rearrangement. Frontiers in Cell and Developmental Biology, 8, 605713.

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Isolation and Characterization of Microglia

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Mice were deeply anesthetized with a lethal dose of choral hydrate and transcardially perfused with 0.1 M PBS. Brain was removed and one forebrain hemisphere (without olfactory bulb) was minced, incubated in phenol-red free DMEM supplemented with 2% heat inactivated FBS, 10mM HEPES and Collagenase type IV (0.4 mg/mL) for 15 min and then passed through a 19G blunt syringe to obtain a homogeneous cell suspension. Mononuclear cells were separated with a 40% Percoll gradient. Isolated cells were surface stained in FACS buffer for 20–30 min on ice with the following antibodies: CD11b (clone M1/70, eBioscience), F4/80 (clone CI: A3-1, BioRad), CD45 (clone 30F11, eBioscience), MHC II (clone M5/114.15.2, eBioscience), CD3e (clone 145-2C11, Biolegend), Gr-1 (clone RB6–8C5, Biolegend), CD115 (clone AFS98, eBioscience). Multiparameter analysis was performed on a LSR II Fortessa (BD) and analyzed with FlowJo software (Tree Star) (Details are included in the Flow Cytometry Reporting Summary). Dead cells and doublets were excluded from all analysis.

Menard C., Pfau M.L., Hodes G.E., Kana V., Wang V.X., Bouchard S., Takahashi A., Flanigan M.E., Aleyasin H., LeClair K.B., Janssen W.G., Labonté B., Parise E.M., Lorsch Z.S., Golden S.A., Heshmati M., Tamminga C., Turecki G., Campbell M., Fayad Z., Tang C.Y., Merad M, & Russo S.J. (2017). Social stress induces neurovascular pathology promoting depression. Nature neuroscience, 20(12), 1752-1760.

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Flow Cytometry Immunophenotyping Protocol

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Flow cytometry was performed exactly as previously described (Basler et al, 2004 (link)). Abs to CD4 (clone GK1.5) and CD45 (clone 30-F11) were obtained from eBioscience and antibodies to CD4 (clone RM4-5) and CD11b (clone M1/70) were purchased from BD Biosciences. Cells were acquired with the use of the Accuri 6 flow cytometer system.

Basler M., Mundt S., Muchamuel T., Moll C., Jiang J., Groettrup M, & Kirk C.J. (2014). Inhibition of the immunoproteasome ameliorates experimental autoimmune encephalomyelitis. EMBO Molecular Medicine, 6(2), 226-238.

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Multiparametric Flow Cytometry of Liver and Spleen

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For flow cytometric analysis, liver and spleen single-cell suspensions were stained using the following antibodies: Cd11c, clone N418 (Biolegend, San Diego, CA, 117339); Ly6c, clone HK1.4 (Biolegend, 128035); MHCII, clone M5/114.15.2 (eBioscience, Waltham, MA, 48-5321-82); Cd45, clone 30-F11 (eBioscience, 56-0451-82); Cd11b, clone M1/70 (eBioscience, 47-0112-82); Cd64, clone X54-5/7.1 (BD Biosciences, Franklin Lakes, NJ, 741024); Ly6g, clone 1A8 (BD Biosciences, 560601); Fc block Cd16/Cd32, clone 2.4G2 (BD Biosciences, 553142); Ly6g, RB6-8c5 (Tonbo, San Diego, CA, 60-5931); Ghost (Tonbo, 13-0870-T100); Flow cytometric data was acquired on the BD LSRII Fortessa X20 and analyzed using FlowJo (Franklin Lakes, NJ).

Alkhani A., Levy C.S., Tsui M., Rosenberg K.A., Polovina K., Mattis A.N., Mack M., Van Dyken S., Wang B.M., Maher J.J, & Nijagal A. (2020). Ly6cLo non-classical monocytes promote resolution of rhesus rotavirus-mediated perinatal hepatic inflammation. Scientific Reports, 10, 7165.

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Immune Cell Analysis in Aortic Tissue

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Blood counts were analyzed using an automated hematology analyzer (KX-21N, Sysmex, Germany). For FACS analyses, tissues were passed through a 70 μm filter (BD Biosciences, Germany) to obtain single-cell suspensions. Whole blood was combined with a red blood cell lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA) to allow the isolation of leukocytes. Whole aortae and aortic roots were excised, flushed with PBS, and enzymatically dissociated using Liberase Blendzyme TL (Roche, Germany) solution for 30 minutes at 37°C. The resulting single cell suspensions were resuspended in Hanks Buffered Saline Solution (HBSS), enumerated using a Neubauer counting chamber. Cells were stained for 30 minutes on ice using combinations of specific antibodies from BD biosciences (CD45, clone 30-F11; CD3, clone 500A2; CD8a, clone 53-6.7; IFNγ, clone XMG1.2) and eBioscience (TCRβ, clone H57-597; CD44, clone IM7; CD62L, clone MEL-14; CD4, clone RM4-5; Foxp3, clone FJK-16s; CD25, clone PC61.5; IL-17a, clone eBio17B7). Intracellular labelling of IL17A and IFNγ was performed using the BD Cytofix/Cytoperm Kit (BD Biosciences). Intracellular labeling of Foxp3 was performed using the Foxp3 Staining Buffer Set (eBioscience) according to the manufacturer's instructions. Probes were analyzed using a FACSCanto II (Becton Dickson, USA) and FlowJo 7.6 software (Treestar Inc., USA).

Cochain C., Chaudhari S.M., Koch M., Wiendl H., Eckstein H.H, & Zernecke A. (2014). Programmed Cell Death-1 Deficiency Exacerbates T Cell Activation and Atherogenesis despite Expansion of Regulatory T Cells in Atherosclerosis-Prone Mice. PLoS ONE, 9(4), e93280.

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Murine Epidermal Cell Isolation and Flow Cytometry

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7 million murine epidermal cells were isolated and cultured with Alexa546-labeled nanoparticles as described previously⁴³. After 20 h samples were stained using antibodies against MHC-class II (clone M5/114.15.2, eBioscience), CD40 (clone 3/23, Becton Dickinson) and CD45 (clone 30-F11, eBioscience) and analyzed on a FACSCantoII™ flow cytometer (Becton Dickinson). Dead cells were excluded by co-staining with 7AAD (Becton Dickinson). About 1 % of the MHC-class II positive cells were dead in the nanoparticle treated samples, and about 0.7% in the untreated controls. A minimum of 200,000 events was acquired for each measurement. FACS data were analyzed with FlowJo software.
For gene expression experiments six hours after skin preparation and nanoparticle administration, the mice were sacrificed, the skin was removed and cultured as described previously. The cells that migrated out from the skin into the culture media were harvested and the large cells containing the LC population were analyzed using flow cytometry.

Tőke E.R., Lőrincz O., Csiszovszki Z., Somogyi E., Felföldi G., Molnár L., Szipőcs R., Kolonics A., Malissen B., Lori F., Trocio J., Bakare N., Horkay F., Romani N., Tripp C.H., Stoitzner P, & Lisziewicz J. (2014). Exploitation of Langerhans cells for in vivo DNA vaccine delivery into the lymph nodes. Gene therapy, 21(6), 566-574.

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Popliteal Lymph Node Single Cell Analysis

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Single cell suspensions of popliteal lymph nodes were prepared by homogenization in Kontes pestle tubes prior to filtration through 70 μm nylon cell strainers. Cells were stained with the following antibodies: CD45 (clone 30-F11, eBioscience, Lot#E10032–1635); CD8α (clone 53–6.7, eBioscience, many different lots); CD69 (clone H1.2F3, eBioscience, Lot#B207773); and IFN-γ (clone XMG1.2, eBioscience, Lot#E024667). Cells were analyzed on an LSR II flow cytometer (BD Biosciences) using FacsDiva software, and resultant data analyzed using FlowJo (Treestar).

Reynoso G.V., Weisberg A.S., Shannon J.P., McManus D.T., Shores L., Americo J.L., Stan R.V., Yewdell J.W, & Hickman H.D. (2019). Lymph Node Conduits Transport Virions for Rapid T Cell Activation. Nature immunology, 20(5), 602-612.

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Kit Expression Analysis in Mouse Tumors

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Flow cytometric analysis was performed on Kit^V558del/+ mouse tumors as before (28 (link)). All cells were analyzed on a FACSAria (BD Biosciences). Mouse-specific antibodies included CD45 (clone 30-F11) and Met (eBioclone 7) from eBioscience, and Kit (CD117; 2B8), Rat IgG2aκ isotype (R35-95), and rat IgG1κ isotype (R3-34) from BD Biosciences.

Cohen N.A., Zeng S., Seifert A.M., Kim T.S., Sorenson E.C., Greer J.B., Beckman M.J., Santamaria-Barria J.A., Crawley M.H., Green B.L., Rossi F., Besmer P., Antonescu C.R, & DeMatteo R.P. (2015). Pharmacological inhibition of KIT activates MET signaling in gastrointestinal stromal tumors. Cancer research, 75(10), 2061-2070.

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