Cd45 biotin

Manufactured by Thermo Fisher Scientific

Sourced in United States

CD45-biotin is a cell surface marker that is commonly used in flow cytometry applications. It is a biotinylated antibody that specifically binds to the CD45 antigen, which is expressed on the surface of most hematopoietic cells. This product can be used to identify and isolate CD45-positive cells from a heterogeneous cell population.

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Anti-CD45.2-biotin (104; (2 citations) CD45-biotin (clone 30-F11, (2 citations) Biotin‐conjugated rat antibodies specific for mouse CD45 (2 citations) Biotin-conjugated anti-CD45 antibody (2 citations)

4 protocols using cd45 biotin

Lung Cell Isolation and FACS Analysis

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Lung cell suspensions were prepared using an elastase digestion and stained for fluorescence-activated cell sorting (FACS), as previously described (20 (link)). Briefly, cells were resuspended in Hanks' balanced saline solution buffer supplemented with 2% fetal bovine serum, 0.1 mM EDTA, 10 mM HEPES, 100 IU/ml penicillin and 100 µg/ml streptomycin (HBSS⁺). Cells were then stained with the primary antibodies on ice for 45 min. The following antibodies were employed: EpCAM-PE-Cy7 (25-5791-80, 1:100), CD31-Biotin (13-0311-81, 1:40), CD34-Biotin (13-0341-81, 1:10), CD45-Biotin (13-0451-81, 1:100), Sca-1-APC (17-5981-81, 1:100), and CD24-PE (12-0242-81, 1:20) (all from eBioscience, Inc., San Diego, CA, USA). Cells were subsequently stained with the secondary antibody on ice for 40 min using streptavidin-APC-Cy7 (47-4317-82, 1:100; eBioscience, Inc.). Dead cells were identified using 7-aminoactinomycin D staining (BD Biosciences, Franlkin Lakes, NJ, USA).

Li X., Yang L., Sun X., Wu J., Li Y., Zhang Q., Zhang Y., Li K., Wu Q, & Chen H. (2017). The role of TGFβ-HGF-Smad4 axis in regulating the proliferation of mouse airway progenitor cells. Molecular Medicine Reports, 16(6), 8155-8163.

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Isolation and Sorting of Lung Immune Cells

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Lung cells were blocked with Fc block (BD Biosciences) for 20 min 4°C and subsequently incubated with CD45-biotin (eBioscience). Cells were washed with FACS buffer, incubated with anti-biotin microbeads (Miltenyi Biotech, San Diego, CA) and run through magnetic columns per manufacture’s protocol. The CD45^pos fraction was collected for RNA. The CD45^neg flow through was stained with CD326-FITC, CD31-PE (eBioscience), and CD45-APC-Cy7 (BD Biosciences) antibodies. Cells were sorted using an Aria cell sorter (BD Biosciences).
For sorting of macrophage and DC populations, lung cells were blocked with Fc block and incubated with antibodies to CD45, MHCII, Siglec F, Ly6G, CD11b, CD11c, CD103. Cells were then washed twice with FACS buffer and sorted directly into 500μl RA1 Lysis Buffer (Clontech) on an Aria cell sorter (BD). Cell populations were defined by the following surface expression patterns. “CD103+ DCs” were defined as CD45⁺/Ly6G⁻/SigF⁻/CD11c⁺/CD103⁺, aAMs” as CD45⁺/Ly6G⁻/SigF^int/CD11c^int/CD11b^high, and “mAMs” were defined as CD45⁺/Ly6G⁻/SigF^high/CD11c^high/CD11b^low, and “CD11b DCs” defined as CD45⁺/Ly6G⁻/CD11c⁺/CD103^neg/CD11b^high/MHCII^high.

Eddy W.E., Gong K.Q., Bell B., Parks W.C., Ziegler S.F, & Manicone A.M. (2017). Stat5 is required for CD103+ Dendritic Cell and Alveolar Macrophage Development and Protection from Lung Injury. Journal of immunology (Baltimore, Md. : 1950), 198(12), 4813-4822.

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Isolation and Characterization of Mouse Lung Cell Types

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A single-cell suspension was prepared by digestion of lung tissues from 6–10-week-old C57BL/6 mice with elastase and DNase I. Freshly isolated cells were suspended in Hanks balanced saline
solution (Solarbio, Beijing, China) with 2% fetal bovine serum (FBS, Gibco), 0.01% Penicillin- streptomycin (Gibco, USA) and 10 mM HEPES (Sigma). The primary antibodies used to stain cells
included CD24-PE (1:25, eBioscience, San Diego, CA, USA), EpCAM-PE-Cy7 (1:50, Biolegend, San Diego, CA, USA), CD31-biotin (1:50, eBioscience), CD45-biotin (1:100, eBioscience), CD34-biotin
(1:16, eBioscience), and Sca-1-APC (1:100, eBioscience). Then, the secondary antibody Streptavidin-APC-Cy7 (1:100, eBioscience) was used, followed by the addition of 7-amino-actinomycin D
(7-AAD, 1:20, eBioscience) to discriminate the dead cells. Mouse club and AT2 cells were sorted based on their surface expression pattern,
CD31⁻CD34⁻CD45⁻EpCAM⁺CD24⁺Sca-1⁺ and
CD31⁻CD34⁻CD45⁻EpCAM⁺CD24⁻Sca-1⁻, respectively.

Zhao F., Wang J., Wang Q., Hou Z., Zhang Y., Li X., Wu Q, & Chen H. (2022). Organoid technology and lung injury mouse models evaluating effects of hydroxychloroquine on lung epithelial regeneration. Experimental Animals, 71(3), 316-328.

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Immunophenotyping and Sorting of Endothelial Cells

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After pre-incubation with Ultra-LEAF purified anti-mouse CD16/32 Ab (Biolegend, San Diego, CA, USA) for 5 min on ice to block of Fc receptors, cells were incubated with primary Abs on ice for 45 min as follows: CD31-biotin (1:50, clone: 390, eBioscience, San Diego, CA, USA), CD45-biotin (1:100, clone: 30-F11, eBioscience) and CD324 (E-Cadherin )-PerCP-eFluor 710 (1:20, Thermo Fisher Scientific). After washing 3 times, cells were incubated with Streptavidin Allophycocyanin (1:200, APC, BD Biosciences, San Jose, CA, USA) secondary Ab for 30 min on ice. After washing 3 times, APC-negative, PerCP-eFluor 710-positive, and tdTomato-positive cells were sorted with a FACSAria cell sorter (BD Biosciences) in preparation for scRNA-seq. Cell viability following FACS was measured using trypan blue vital dye staining, and >95% of cells were confirmed to be viable. To confirm tdTomato expression in sorted cells, 1 × 10⁵ sorted cells were fixed with 4% paraformaldehyde (PFA), and cytospins were prepared by applying cells to EZ Single Cytofunnels (Thermo Fisher Scientific). Cytospins were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA), and tdTomato expression was confirmed using a Nikon Eclipse 80i microscope (Nikon Instruments, Inc., Melville, NY, USA).

Horie M., Castaldi A., Sunohara M., Wang H., Ji Y., Liu Y., Li F., Wilkinson T.A., Hung L., Shen H., Kage H., Offringa I.A., Marconett C.N., Flodby P., Zhou B, & Borok Z. (2020). Integrated Single-Cell RNA-Sequencing Analysis of Aquaporin 5-Expressing Mouse Lung Epithelial Cells Identifies GPRC5A as a Novel Validated Type I Cell Surface Marker. Cells, 9(11), 2460.

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