Cd45 alexa fluor 700

Manufactured by Thermo Fisher Scientific

CD45-Alexa Fluor 700 is a fluorescently labeled antibody that binds to the CD45 antigen. CD45 is a transmembrane protein tyrosine phosphatase that is expressed on the surface of hematopoietic cells. The Alexa Fluor 700 fluorescent dye is conjugated to the antibody, allowing for detection and analysis of CD45-positive cells using flow cytometry or other fluorescence-based techniques.

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Anti-mouse CD45 Alexa Fluor® 700 Alexa Fluor 700 Alexa Fluors

4 protocols using cd45 alexa fluor 700

Multiparameter Tumor Immune Profiling

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Single cell suspensions of collagenase-digested tumors were stained with the following antibodies purchased from eBioscience: fixable viability dye efluor 450, CD69-FITC, PD-1-PE, CD4-PeCy7, CD45-Alexa Fluor 700, CD8a-PE efluor 610, CD40-FITC, CD70-PE, MHC-II IAd-APC, CD11c-PE efluor 610, CD45-APC, CD3-PerCP-Cy5.5, CD11b-PerCP-Cy5.5, EpCAM-PeCy7, PD-L1-PE and PD-L2-FITC. Phospho-Smad2/3 levels were assessed as previously described (27 (link)). Briefly, TDLN cells were stained with anti-mouse CD4-PE and anti-mouse CD8-FITC (eBioscience), fixed, permeabilized (Foxp3 Fixation/Permeabilization Concentrate and Diluent kit, eBioscience) and stained with goat anti-phospho-Smad2/3 (Ser 423/425) followed by APC-labeled donkey anti-goat IgG (Santa Cruz Biotechnology). All samples were acquired with LSRII flow cytometer and analyzed with FlowJo software (version 7.3.6).

Vanpouille-Box C., Diamond J.M., Pilones K.A., Zavadil J., Babb J.S., Formenti S.C., Barcellos-Hoff M.H, & Demaria S. (2015). TGFβ is a master regulator of radiation therapy-induced anti-tumor immunity. Cancer research, 75(11), 2232-2242.

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Multiparameter Flow Cytometry Analysis

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Single-cell suspension cells were incubated with the respective conjugated antibody for 15 min at 4 °C and analyzed with a BD LSR. IgM-APC (catalog: 551062) was from BD Biosciences. CD19-FITC (catalog: 11-0199), CD45-Alexa Fluor 700 (catalog: 56-9459), CD3-PerCP-Cy5.5 (catalog: 45-0037) and CD34-PE-Cyanine 7 (catalog: 25-0349) were from eBioscience. CD38- PerCP-Cy5.5 (catalog: B49199), TDT-PITC (catalog: IM3524), CD22-PE (catalog: IM1835U), CD5-PerCP-Cyanine 5.5 (catalog: B49191), CD19-ECD (catalog: 652804), CD10-PE (catalog: A07760) and CD34-ECD (catalog: IM2709U) were from Beckman Coulter. Propidium iodide were from Sigma. The gating strategy for MCL cells were selected using CD19^–/IgM^–, CD19^–/IgM⁺ and CD19⁺/IgM⁺cells (gate i: CD45⁺/PI^–; gate ii: CD34^–/CD3^–; gate iii: CD19^–/IgM^–, CD19^–/IgM⁺ and CD19⁺/IgM⁺). The sorting purity was greater than 99% in the majority of samples. All the fractions were isolated by fluorescence-activated cell sorting (Aria, Becton Dickinson, San Jose, CA).

Tang J., Zhang L., Zhou T., Sun Z., Kong L., Jing L., Xing H., Wu H., Liu Y., Zhou S., Li J., Chen M., Xu F., Tang J., Ma T., Hu M., Liu D., Guo J., Zhu X., Chen Y., Ye T., Wang J., Li X, & Xing H.R. (2018). Identification and characterization of the cellular subclones that contribute to the pathogenesis of mantle cell lymphoma. Genes & Diseases, 6(4), 407-418.

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Isolation and Characterization of Mouse PVAT

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Mouse PVAT was dissected from the thoracic aorta as previously described.(Chang et al., 2012) To avoid the effects of the adventitia, only lipid droplet positive tissue was collected. Adipose tissue was cut into small pieces and then digested with 0.2% collagenase (Sigma‐Aldrich) at 37°C for 45 min. The cell suspension was filtered through 70‐µm nylon cell strainer (Falcon) twice to remove tissue debris. The stromal cells were resuspended in red blood cell lysis buffer (eBioscience) for 10 min and then were maintained in 1% bovine serum albumin (BSA) in PBS containing fluorochrome‐conjugated antibodies directed against the following cell surface markers: CD45‐Alexa Fluor700, Lin‐eFluor450, Sca1‐PE‐Cy7, CD90‐PE, cKit‐APC, CD34‐FITC (eBioscience). Flow cytometry was performed on a BD flow cytometer (Verse). FCS files were exported and analyzed using FlowJo 8.3.3 software (Tree Star Inc).

Pan X., Ruan C., Liu X., Kong L., Ma Y., Wu Q., Li H., Sun Y., Chen A., Zhao Q., Wu F., Wang X., Wang J., Zhu D, & Gao P. (2019). Perivascular adipose tissue‐derived stromal cells contribute to vascular remodeling during aging. Aging Cell, 18(4), e12969.

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Characterization of Cryopreserved CLL PBMCs

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Cryopreserved PBMCs from 8 CLL patients with increased absolute lymphocyte count (ALC) levels (≥20x10 9 cells/L) were further characterized. Cells were thawed and labeled with DAPI and with the following surface antibodies: CD45 (Alexa Fluor 700, eBioscience), CD19 (FITC, BD Biosciences), CD5 (PerCP-Cy5.5, BD Biosciences), CD20 (APC, BD Biosciences), HLA-E (PE, Miltenyi Biotec) and isotype control (PE, eBioscience). For the analysis of HLA-G expression on tumor cells, unlabeled antibodies against HLA-G (clone G233) (Invitrogen) and MYC (clone 9E10, used as negative control) were employed, followed by indirect immunofluorescence staining with PE-Cy7 goat anti-mouse IgG (minimal x-reactivity) (Biolegend). Data acquisition was performed in a LSR Fortessa instrument (BD Biosciences) and analyzed using FlowJo. Mean fluorescence intensity (MFI) values were evaluated as shown in Supplemental Methods and in Supplemental Figure 3.

Puiggros A., Blanco G., Muntasell A., Rodríguez-Rivera M., Nonell L., Altadill M., Puigdecanet E., Arnal M., Calvo X., Gimeno E., Abella E., Abrisqueta P., Bosch F., Yélamos J., Ferrer A., López-Botet M, & Espinet B. (2021). Reduced expansion of CD94/NKG2C⁺ NK cells in chronic lymphocytic leukemia and CLL-like monoclonal B-cell lymphocytosis is not related to increased human cytomegalovirus seronegativity or NKG2C deletions. International journal of laboratory hematology, 43(5).

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