Cd45 30 f11

Manufactured by Thermo Fisher Scientific

Sourced in Germany, United States

The CD45 (30-F11) is a laboratory reagent used for the detection and analysis of CD45-expressing cells, such as lymphocytes, in biological samples. It is a monoclonal antibody that binds specifically to the CD45 cell surface antigen. The CD45 antigen is a receptor-linked protein tyrosine phosphatase that plays a crucial role in the regulation of T and B cell antigen receptor signaling.

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Anti-CD45 (clone 30-F11, (23 citations) Anti-CD45 (30-F11, (15 citations) APC-Cy7-anti-CD45 (clone 30-F11), (3 citations) APC-conjugated CD45 (30-F11; (2 citations) Anti-CD45-PE (clone 30-F11 (2 citations) FITC-conjugated anti–mouse CD45 (30-F11; (2 citations) Anti-CD45-eF450 (clone 30-F11; (2 citations) EFluor 450-conjugated anti-mouse CD45 (clone 30-F11, (2 citations) CD45-Percp-Cy7 (clone 30-F11, (2 citations) Anti-CD45 APC (30-F11; (2 citations)

34 protocols using cd45 30 f11

Isolation and Analysis of Lymphoid and Splenic Cells

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LNs, and spleens where indicated, were incubated in digestion medium (RPMI, 0.1 mg/ml DNase I (Invitrogen), 0.1–0.2 mg/ml liberase (Roche) and 0.8 mg/ml dispase (Roche)^{19 (link)}. Collected single-cell suspensions were filtered. Spleen samples underwent red blood cell lysis. For flow cytometry, cells were stained with Ghostdye510 viability dye (Tonbo Biosciences) in PBS, followed by labeling with CD45 (30-F11, Invitrogen), CD45.2 (104, BD Biosciences), PDPN (8.1.1, Biolegend), CD31 (MEC 13.3, BD Biosciences) and Ki67 (SolA15, eBioscience), Madcam-1 (MECA-89, BD), CD21/CD35 (Clone: 7G6, BD), Cpt1a (8F6AE9, Abcam), CD4 (GK1.5, Invitrogen), B220 (RA3–6B2, BD), IL-17RA (PAJ-17R, Invitrogen) and CountBright™ Absolute Counting Beads (Molecular Probes). For detection of apoptosis, FRC were stained with FITC Active Caspase-3 Apoptosis Kit (BD Biosciences), as per manufacturer’s protocol. For in vivo cell cycle analysis, mice were injected i.p. with 1 mg of bromodeoxyuridine (BrdU flow kit; BD Biosciences) 24 h before sacrifice. Data acquired with a FACS Fortessa (BD Biosciences), analyzed using FlowJo (Tree Star).

Majumder S., Amatya N., Revu S., Jawale C.V., Wu D., Rittenhouse N., Menk A., Kupul S., Du F., Raphael I., Bhattacharjee A., Siebenlist U., Hand T.W., Delgoffe G.M., Poholek A.C., Gaffen S.L., Biswas P.S, & McGeachy M.J. (2019). IL-17 metabolically reprograms activated fibroblastic reticular cells for proliferation and survival. Nature immunology, 20(5), 534-545.

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Multiparametric Flow Cytometry Analysis

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Cells were pre-incubated with a FcγR-blocking mAb (CD16/32; 2.4G2, BD Bioscience) for 20 min followed by incubation with specific mAbs for 20 min on ice. After staining surface molecules, the cells were re-suspended in fixation/permeabilization solution (eBioscience), and intracellular staining of FOXP3 was performed with a FOXP3 staining buffer kit (eBioscience). Flow cytometric analyses were performed on a LSR II system (Becton Dickinson), and data were analyzed using Flowjo software (Tree Star). Background fluorescence was assessed by staining with isotype-matched control mAbs. FITC-, PE-, PerCP-Cy5.5-, APC-, PE-Cy7-, APC-Cy7- or AlexaFluor 647-conjugated mAbs against CD4 (GK1.5), CD3 (145-2C11), CD11b (M1/70), CD11c (N148), Ly6c (HK1.4), MHCII (M5/114.15.2), 7AAD and FOXP3 (FJK-16S) were from eBioscience, CD45 (30-F11) and Ly6G (1A8) were from Invitrogen.

Lee Y., Sugihara K., Gillilland MG I.I.I., Jon S., Kamada N, & Moon J.J. (2019). Hyaluronic acid-bilirubin nanomedicine for targeted modulation of dysregulated intestinal barrier, microbiome, and immune responses in colitis.. Nature materials, 19(1), 118-126.

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Comprehensive Immune Cell Profiling

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For cell staining and flow cytometry, fluorochrome-conjugated antibodies to CD3 (clone S4.1; Invitrogen), CD4 (S3.5; Invitrogen), CD8 (RPA-T8; BioLegend), CD16 (3G8; BD Biosciences), CD45 (30-F11; Invitrogen), CD56 (NCAM16.2; BD Biosciences), CD57 (HCD57; BioLegend), CD107a (H4A3; BD Biosciences), perforin (δG9; BD Biosciences), granzyme A (CB9; BD Biosciences), and granzyme B (GB11; BD Biosciences), were used. Fluorochrome-conjugated IgG1 and IgG2b (MOPC-21 and 27-35; BD Biosciences) isotype control antibodies were also used in addition to a fixable live/dead cell marker (Invitrogen). Mouse anti-CD3 (S4.1) and purified anti-CD16 (3G8) mAbs were used for redirected ADCC. For confocal microscopy, mouse monoclonal antibodies to α-tubulin (236-10501; Invitrogen), mannose-6-phosphate receptor (MEM-238; Invitrogen), and perforin (δG9; BioLegend) mAbs were used. Rabbit polyclonal antibodies used were Rab27a, Stx11, and Munc13-4 (all Protein Technologies Group), EEA1 (Cell Signaling Technologies), WASP-interacting protein (WIP, Santa-Cruz), and Cathepsin D (Upstate). Secondary antibodies were donkey anti-mouse and donkey anti-rabbit (both Invitrogen). DNA was labeled with DAPI and actin with phalloidin (both Invitrogen).

Chiang S.C., Wood S.M., Tesi B., Akar H.H., Al-Herz W., Ammann S., Belen F.B., Caliskan U., Kaya Z., Lehmberg K., Patiroglu T., Tokgoz H., Ünüvar A., Introne W.J., Henter J.I., Nordenskjöld M., Ljunggren H.G., Meeths M., Ehl S., Krzewski K, & Bryceson Y.T. (2017). Differences in Granule Morphology yet Equally Impaired Exocytosis among Cytotoxic T Cells and NK Cells from Chediak–Higashi Syndrome Patients. Frontiers in Immunology, 8, 426.

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Multiparametric Flow Cytometry Analysis

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Multiparametric Flow Cytometry Analysis

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Monoclonal antibodies used were EpCAM-1 (G8.8; BioLegend); CD45 (30F11; Invitrogen); Lin-biotin (eBioscience); CD19 (1D3), CD3 (145-2C11), CD11c (N418), CD11b (M1/70), Gr1 (RB6-8C5), and Streptavidin (BioLegend); NK1.1 (PK136; eBioscience); CD127 (A7R34; eBioscience); CD117 (2B8; BD); NKp46 (2941.4; eBioscience); and CCR6 (29-2L17; BioLegend).

Aparicio-Domingo P., Romera-Hernandez M., Karrich J.J., Cornelissen F., Papazian N., Lindenbergh-Kortleve D.J., Butler J.A., Boon L., Coles M.C., Samsom J.N, & Cupedo T. (2015). Type 3 innate lymphoid cells maintain intestinal epithelial stem cells after tissue damage. The Journal of Experimental Medicine, 212(11), 1783-1791.

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Tumor Immune Cell Isolation

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The tumors were harvested and digested with collagenase IV. The resulting cells were lysed with red blood cell lysis buffer (Beyotime, China) and passed through nylon mesh filters (70 μm). To analyze the T-cells, single-cell suspensions were resuspended in PBS labeling solution with CD45 (30-F11, Invitrogen), CD3 (17A2, BD), and CD8 (53–6.7, BD). CD11b (M1/70, Invitrogen) and Gr-1 (RB6–8C5, Invitrogen) were labeled single cells for MDSCs.

Liu H., Han J., Lv Y., Zhao Z., Zheng S., Sun Y, & Sun T. (2023). Isorhamnetin and anti-PD-L1 antibody dual-functional mesoporous silica nanoparticles improve tumor immune microenvironment and inhibit YY1-mediated tumor progression. Journal of Nanobiotechnology, 21, 208.

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Tracking Chimerism in Recipient Fetuses

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Recipient fetuses were tracked for chimerism in peripheral blood (PB) serially starting at three weeks of age. LDMCs were isolated as described above. Antibodies used for determining chimerism were: CD45 (30-F11, eBioscience), H-2 Kd (SF1-1.1, BD Pharmingen) or H-2Dd (34-2-12), and H-2Kb (AF6-88.5, eBioscience). Dead cells were excluded using Hoechst 33528 (Invitrogen). Percentage non-erythroid donor chimerism was determined by the percentage of total CD45+ events that express donor MHC class I (H-2 Kd or H-2Dd in Balb/c → B6 or Balb/c → B6.CD1d^−/− chimeras, H-2Kb in B6 → Balb/c chimeras).

Strong B.S., Newkold T.J., Lee A.E., Turner L.E., Alhajjat A.M., Heusel J.W, & Shaaban A.F. (2016). Extrinsic allospecific signals of hematopoietic origin dictate iNKT cell lineage-fate decisions during development. Scientific Reports, 6, 28837.

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Multiparametric Flow Cytometry of Immune Cells

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Single cell suspensions were incubated with an anti-FcγIII/II receptor antibody (clone 93) to block unspecific binding and Zombie Violet^TM (Biolegend, Germany), a fixable viability dye. Thereafter, cells were stained with fluorochrome conjugated antibodies against cell surface markers: CD45 (30-F11, eBioscience, Germany), CD11b (M1/70, eBioscience, Germany), Ly6G (1A8, BD Biosciences, Germany), Ly6C (HK1.4, eBioscience, Germany), CCR2 (475301, R&D, USA), TREM2 (237920, R&D, USA), CD36 (HM36, Biolegend, Germany), in FACS buffer (PBS containing 2 % FCS and 0.1 % NaN₃) for 30 min on ice and then washed and fixed in 4 % paraformaldehyde (PFA) for 10 min.

Möhle L., Israel N., Paarmann K., Krohn M., Pietkiewicz S., Müller A., Lavrik I.N., Buguliskis J.S., Schott B.H., Schlüter D., Gundelfinger E.D., Montag D., Seifert U., Pahnke J, & Dunay I.R. (2016). Chronic Toxoplasma gondii infection enhances β-amyloid phagocytosis and clearance by recruited monocytes. Acta Neuropathologica Communications, 4, 25.

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Spleen and Heart Cell Isolation and Characterization

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Single cell suspensions from spleens and lymph nodes were obtained via passing through 70 μm cell strainers in cold PBS^−/−. Hearts were collected and digested with Liberase TM (Roche). Erythrocytes were removed with lysis buffer (BD Biosciences) from spleen and heart cell suspensions. Cells were stained at the following dilutions of stock reagents: Live/dead Aqua Fluorescent Reactive Dye 1:1,000 (Life Techonologies), anti-mouse CD16/32 1:100 (2.4G2, BD Pharmigen), anti-mouse 1:100 CD45 (30-F11, eBioscience), anti-mouse 1:100 CD3ɛ (145-2C11, BioLegend), anti-mouse 1:100 CD19 (eBio1D3, eBioscience), anti-mouse 1:100 CD11b (M1/70, Biolegend), 1:100 CD11c (Bu15, eBioscience), F4/80 1:100 (CI:A3-1, Serotec) in Supplementary Fig. 5d, F4/80 1:100 (BM8, eBioscience) in Fig. 5d,e and Supplementary Fig. 5c, IL-10 1:80 (JES5-16E3, eBioscience), FoxP3 1:100 (FJK-165, eBioscience), Ly6C 1:100 (HK1.4, eBioscience) or anti-CD25 1:100 (PC61.5, eBioscience). An eBioscience intracellular staining kit was used were applicable. Samples were acquired on a fluorescence-activated cell sorting Canto II (BD) and analysed with FlowJo10.

Kallikourdis M., Martini E., Carullo P., Sardi C., Roselli G., Greco C.M., Vignali D., Riva F., Ormbostad Berre A.M., Stølen T.O., Fumero A., Faggian G., Di Pasquale E., Elia L., Rumio C., Catalucci D., Papait R, & Condorelli G. (2017). T cell costimulation blockade blunts pressure overload-induced heart failure. Nature Communications, 8, 14680.

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Isolation of Immune Cells from Murine Ears

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To prepare single cell suspension, ventral and dorsal sheets of the ears were separated from the cartilage and incubated for 90 min in CO2 incubator at 37°C in 1 mL volume of RPMI 1640 (Sigma-Aldrich, #R7388) containing 0.25 mg ml⁻¹ Liberase TL (Roche Diagnostics, #5401020001). The digested ears were passed through a 3 mL syringe to make single-cell suspension. The cells were filtered through 70 μm nylon mesh and washed in FACS buffer at 1500 rpm for 5 minutes. Cells were suspended in FACS buffer for further analysis. For surface staining the following antibodies were used at 1:100 dilutions in FACS buffer according to the manufacture’s specifications. CD45 (30-F11, eBiosciences), CD3 (17A2, eBiosciences), CD90.2 (53–2.1, eBiosciences), βTCR (H57–597, eBiosciences), CD4 (RM4–5, Biolegend), CD8 (YTS5167.7, eBiosciences), CD11b (M1/70, eBiosciences), Ly6G (1A8, eBiosciences), and Ly6C (AL-21, BD Pharmingen). For counting the cells AccuCount Fluorecent particles (Spherotech) were used. The stained cells were run on BD FACSymphony^™A3 (BD Biosciences) and the acquired data were analyzed using FlowJo software (Tree Star).

Ni C.Z., Welsh K., Leo E., Chiou C.K., Wu H., Reed J.C, & Ely K.R. (2000). Molecular basis for CD40 signaling mediated by TRAF3. Proceedings of the National Academy of Sciences of the United States of America, 97(19), 10395-10399.

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