Alexa fluor 488 anti cd45

Manufactured by BioLegend

Sourced in United States

Alexa Fluor 488 anti-CD45 is a fluorescently labeled antibody that binds to the CD45 antigen. CD45 is a transmembrane protein tyrosine phosphatase expressed on the surface of most hematopoietic cells. The Alexa Fluor 488 dye is used to label the antibody, providing a green fluorescent signal that can be detected by flow cytometry or other fluorescence-based applications.

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Alexa Fluor 488-conjugated anti-CD45 Alexa Fluor-488 anti-mouse CD45.1 antibody Anti-mouse CD45.2-Alexa Fluor 488 (clone 104 Alexa Fluor 488 anti-mouse CD45 antibody Anti-CD45–Alexa Fluor 488 (clone 30-F11; Alexa Fluor-488 anti-mouse CD45.2 antibody Alexa Fluor 488 Alexa 488 Anti-CD45

7 protocols using alexa fluor 488 anti cd45

Comprehensive Immune Cell Profiling of CNS

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Cells were suspended in 1 ml of FACS buffer (PBS with 2% FBS). After Fc receptor blocking with antimouse CD16/32 (BioLegend), cells were incubated with a mix of antibodies specific to analysis at 1:200 dilution each for 20 min, and dead cell marker 7-aminoactinomycin D (5 µl/sample) or DAPI (1 µl/sample) was added right before acquisition on a BD FACSCanto II device. Data were analyzed using BD FACSDiva software. Cell counts were reported to initial cell number determined after Percoll density gradient purification.
The antibody panel for CNS-infiltrating cells was as follows: Alexa Fluor 488 anti-CD45, APC anti-CD3, PE anti-CD11b, PE–cyanine 7 anti-F4/80, and 7- aminoactinomycin D (BioLegend). The antibody panel for RNAseq was as follows: Alexa Fluor 488 anti-CD45, APC anti-CD3, PE anti-CD11b, and DAPI (BioLegend).

Mimouna S., Rollins D.A., Shibu G., Tharmalingam B., Deochand D.K., Chen X., Oliver D., Chinenov Y, & Rogatsky I. (2020). Transcription cofactor GRIP1 differentially affects myeloid cell–driven neuroinflammation and response to IFN-β therapy. The Journal of Experimental Medicine, 218(1), e20192386.

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Multicolor Immunofluorescent Staining Protocol

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The protocol for immunohistochemical staining was as described previously^{30 (link)}. Staining reagents included biotinylated PNA (Vector), AlexaFluor 647 anti-B220, biotinylated anti-IgD, AlexaFluor 488 anti-CD45.2 (Biolegend), and streptavidin AlexaFluor 568 (Invitrogen). All stained slides were mounted with ProlongGold antifade reagents (Invitrogen) and examined with an Olympus FV1000 confocal system.

Liu X., Chen X., Zhong B., Wang A., Wang X., Chu F., Nurieva R.I., Yan X., Chen P., van der Flier L.G., Nakatsukasa H., Neelapu S.S., Chen W., Clevers H., Tian Q., Qi H., Wei L, & Dong C. (2014). Transcription factor Achaete-Scute homologue 2 initiates T follicular helper cell development. Nature, 507(7493), 513-518.

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Multicolor Immunofluorescent Staining Protocol

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Multiparametric Flow Cytometry of Immune Cells

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BALF cells, mLN, or lung single cell suspensions were resuspended in cell staining buffer (no. 420201, BioLegend, San Diego, CA, USA) and blocked with TruStain FcX (anti-mouse CD16/32) antibody (no. 101320, BioLegend, San Diego, CA, USA) for 10–15 min on ice. The cells were then incubated 30 min with the following specific fluorochrome-conjugated antibodies or isotype controls (BioLegend, San Diego, CA, USA ) in the dark at 4 °C: Alexa Fluor-488 anti-CD45, PE anti-CD3, APC anti-CD4, FITC anti-CD4, PE-Cy7 anti-CD8α, APC anti-glycosylated CD43 (1B11), FITC anti-CD43, FITC anti-CD44, APC anti-CD11a, PE/Cy5 anti-CD69, Pacific Blue anti-CD54, APC anti-CD11b, PE anti-Ly6G, or FITC isotype control antibodies. PE-conjugated tetramer specific for H-2Db IAV NP_366–374 (ASNENMETM) was from MBL International Corporation (Woburn, MA, USA). After washing, stained cells were acquired in an Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA USA). Cell death was determined by flow cytometry using APC annexin V apoptosis detection kit with propidium iodide (no. 640932, BioLegend, San Diego, CA, USA). Flow cytometry data were analyzed by using FlowJo software v10.6.2.

Hao Q., Kundu S., Shetty S, & Tang H. (2024). Runx3 Regulates CD8+ T Cell Local Expansion and CD43 Glycosylation in Mice by H1N1 Influenza A Virus Infection. International Journal of Molecular Sciences, 25(8), 4220.

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Immunophenotyping of Mouse Inflammation

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IFNβ was purchased from PBL Interferon Source (Piscataway, NJ), and tPA was purchased from Genentech (San Francisco, CA). Evans blue was purchased from Sigma-Aldrich (St. Louis, MO). Triphenyltetrazolium chloride (TTC) and trichloroacetic acid (TCA) were purchased from Alfa Aesar (Tewksbury, MA). Antibodies of Alexa Fluor 488 anti-CD45 (Clone: 30-F11), FITC anti-CD11a (Clone: M17/4), PE/Cy7 anti-Ly6C (Clone: HK1.4), APC anti-CD11b (Clone: M1/70), PE/Cy7 anti-CD68 (Clone: FA-11), PE/Cy7 anti-CD86 (Clone: GL-1), PE/Cy7 anti-CD206 (Clone: C068C2), APC anti-IFNAR1 (Clone: MAR1-5A3), and PE anti-IL-1α (Clone: ALF-161) were purchased from BioLegend (San Diego, CA). APC anti-Arg1 antibody was purchase from R&D Systems. APC anti-IL-1β (Clone: NJTEN3) antibody was purchase from eBioscience (Waltham, MA).

Kuo P.C., Weng W.T., Scofield B.A., Paraiso H.C., Bojrab P., Kimes B., Yu I.C, & Yen J.H. (2023). Interferon-β modulates microglial polarization to ameliorate delayed tPA-exacerbated brain injury in ischemic stroke. Frontiers in Immunology, 14, 1148069.

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Multiparameter Flow Cytometry Analysis

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BALF cells, thymic and lung single cell-suspensions were used for flow cytometry analyses. Cells were resuspended in cell staining buffer (no. 420201, BioLegend, San Diego, CA) and blocked with TruStain FcX (anti-mouse CD16/32) antibody specific for FcγR III/II (no. 101320, BioLegend) for 10-15 min on ice. Cell surface staining was performed by incubating the cells in the dark for 30 min at 4°C with following specific fluorochrome-conjugated antibodies or isotype controls (BioLegend unless otherwise specified): Alexa Fluor-488 anti-CD45, PE anti-CD3, APC anti-CD4, FITC anti-CD4, PE-Cy7 anti-CD8α, PE anti-F4/80, APC anti-CD11b, Alexa Fluor-488 anti-CD11b, PE/Cy7 anti-CD11c, PE anti-Ly6G, or FITC isotype control antibodies as well as PE-conjugated tetramer specific for H-2Db IAV NP_366-374 (MBL International Corporation, Woburn, MA). After washing, stained cells were acquired in an Attune NxT flow cytometer that can detect up to 14 colors (Thermo Fisher Scientific) and data were analyzed using FlowJo software v10.6.2.

Hao Q., Kundu S., Shetty S., Tucker T.A., Idell S, & Tang H. (2022). Inducible general knockout of Runx3 profoundly reduces pulmonary cytotoxic CD8+ T cells with minimal effect on outcomes in mice following influenza infection. Frontiers in Immunology, 13, 1011922.

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Immune Cell Profiling in Pancreatitis

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All experiments were done with approval from the Weizmann Institute Animal Care and Use Committee. Animals were sacrificed at the age of 10 wk. Blood and pancreata samples were stained with AlexaFluor488 anti-CD45, AlexaFluor647 anti-NK1.1, and AlexaFluor647 anti-CD3 antibodies (Biolegend) and analyzed by FACS or ImageStram. The SA-β-gal and H&E stainings were performed as described (Krizhanovsky et al. 2008 (link)). To detect immune cells in tissue sections from the pancreas, the mice were subjected to a series of seven hourly intraperitoneal injections of Caerulein (50 ng/g per injection) for 2 d in a row to induce pancreatitis. Twenty-four hours after the last injection, mice were sacrificed, and pancreata were taken, fixed in 4% PFA for 5 h at room temperature, cryoprotected in 30% sucrose, and snap-frozen in OCT. Cryosections (10 µm) were then stained with the same antibodies as for FACS.

Biran A., Perelmutter M., Gal H., Burton D.G., Ovadya Y., Vadai E., Geiger T, & Krizhanovsky V. (2015). Senescent cells communicate via intercellular protein transfer. Genes & Development, 29(8), 791-802.

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