Apc cyanine7 anti mouse cd45

Manufactured by BioLegend

Sourced in United States

The APC/Cyanine7 anti-mouse CD45 is a fluorochrome-conjugated antibody that specifically binds to the CD45 antigen expressed on mouse leukocytes. It can be used in flow cytometry applications to identify and distinguish different types of mouse immune cells.

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APC/Cyanine7 anti-mouse CD45 Antibody APC/Cyanine7 anti-mouse CD45 antibody(30-F11, APC/Cyanine7 mouse anti-human CD45 (2D1) Anti-CD45-APC/Cyanine7 APC anti-mouse CD45 Anti-CD45-APC Anti-mouse CD45 CD45-APC Anti-CD45

8 protocols using apc cyanine7 anti mouse cd45

Comprehensive Immunological Profiling of Tumor Cell Lines

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4T1, H22, HepG2, NIH‐3T3, and Panc02 were purchased from BeNa Culture Collection, China. Anti‐mouse PD‐1 antibody (PD‐1 Ab, BE0146‐100 mg) was purchased from Bio X Cell. Anti‐human PD‐1 Ab was obtained as a gift from Livzon Pharmaceutical Group Inc., China. Flow cytometry antibodies included PE anti‐mouse CD274 (B7‐H1, PD‐L1) (124308), APC/Cyanine7 anti‐mouse CD45 (103132), FITC anti‐mouse CD3 (100204), Brilliant Violet 421 anti‐mouse CD4 (100563), PE/Cy7 anti‐mouse CD8a (100722), PE anti‐mouse IFN‐γ (505808), Alexa Fluor 647 anti‐human/mouse Granzyme B (515406), APC anti‐mouse Ki‐67 (652406), PE anti‐mouse/human CD44 (103008), PE anti‐mouse Ly‐6G/Ly‐6C (Gr‐1) (108407), APC/Cyanine7 anti‐mouse/human CD11b (101226), Brilliant Violet 421 anti‐mouse F4/80 (123137), PE anti‐mouse CD86 (105008), PE/Cy7 anti‐mouse CD206 (MMR) (141720), Alexa Fluor 647 anti‐mouse FOXP3 (126408), APC anti‐mouse CD49b (pan‐NK cells) (108910), PE anti‐mouse CD25 (102008), PE anti‐mouse/human CD44 (103008), APC anti‐mouse CD62L (104412), Zombie Aqua Fixable Viability Kit (423102), Cell Activation Cocktail (with Brefeldin A) (423304), True‐Nuclear Transcription Factor Buffer Set (424401), and Intracellular Staining Permeabilization Wash Buffer (10X) (421002) were purchased from BioLegend.

Liu X., Ye N., Liu S., Guan J., Deng Q., Zhang Z., Xiao C., Ding Z., Zhang B., Chen X., Li Z, & Yang X. (2021). Hyperbaric Oxygen Boosts PD‐1 Antibody Delivery and T Cell Infiltration for Augmented Immune Responses Against Solid Tumors. Advanced Science, 8(15), 2100233.

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Profiling Tumor Immune Cells in OX40 Antibody-Treated Mice

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OX40-humanized mice were implanted with MC38 tumor cells and treated with OX40 antibodies on days 9 and 14 post implantation. On day 17, tumors were collected and dissociated into single cell suspensions by using a digestive solution (1640 medium + 2%FBS + Collagenase IV (Sigma, C5138) + DNase I (Sigma, D5025)), and spleens were ground with sterilized glass slides and filtered through a steel mesh. Red blood cells were lysed using red cell lysing buffer (TIANGEN, RT122). Single cell suspensions were first incubated with the LIVE/DEAD™ Fixable Green Dead Cell Stain Kit (Invitrogen, L34970), then labeled with the following antibodies in flow cytometric analyses: Brilliant Violet 421™ anti-mouse CD3 (BioLegend, 100228), PE anti-mouse Ki-67 (BioLegend, 652404), PerCP anti-mouse CD8a (BioLegend, 100732), APC/Cyanine7 anti-mouse CD45 (BioLegend, 103116), PE/Cyanine7 anti-mouse IFN-γ (BioLegend, 505826), eFluor™ 450 anti-FoxP3 (Invitrogen, 48-5773-82) and eFluor™ 506 anti-CD4 (Invitrogen, 69-0042-82). Intracellular FoxP3, IFN-γ and Ki67 were labeled following the product manual. Flow cytometry analysis was performed using Cytek^® Aurora (Cytek Biosciences).

Liang S., Zheng D., Liu X., Mei X., Zhou C., Xiao C., Qin C., Yue H., Lin J., Liu C., Li S, & Yu J.C. (2023). BAT6026, a novel anti-OX40 antibody with enhanced antibody dependent cellular cytotoxicity effect for cancer immunotherapy. Frontiers in Oncology, 13, 1211759.

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Characterization of Mesenchymal Stem Cells

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bMSCs were isolated and expanded, as described, with minor modification.²¹ The cells were characterized phenotypically using cell surface markers (positive for CD29, Scal‐1, CD44, and CD105 and negative for CD34, CD45, CD11b, and CD31) with flow cytometric analysis (Figure S1A) and multilineage differentiation with chemical induction and staining, including adipogenic differentiation using oil red O staining, osteogenic differentiation using alizarin red staining, and chondrogenic differentiation using Alcian blue and nuclear fast red staining (Figure S1B through S1E). The antibodies for flow cytometry analysis, including fluorescein isothiocyanate (FITC) anti‐mouse CD29 (102205), Peridinin‐Chlorophyll‐Protein anti‐mouse Sca‐1 (108122), FITC anti‐mouse CD44 (103005), Allophycocyanin (APC) anti‐mouse CD105 (120413), Phycoerythrin anti‐mouse CD34 (119308), APC/cyanine7 anti‐mouse CD45 (103115), AF 700 anti‐mouse CD11b (101222), and PE/cyanine7 anti‐mouse CD31 (102418), were from Biolegend. Cell populations were carefully compensated with isotype antibody staining as control. Fluorescence‐positive cells were quantitatively evaluated using LSRFortessa X‐20 (BD Bioscience, CA), and analyzed using software FlowJo_V10.

Zhu Q., Hao H., Xu H., Fichman Y., Cui Y., Yang C., Wang M., Mittler R., Hill M.A., Cowan P.J., Zhang G., He X., Zhou S, & Liu Z. (2021). Combination of Antioxidant Enzyme Overexpression and N‐Acetylcysteine Treatment Enhances the Survival of Bone Marrow Mesenchymal Stromal Cells in Ischemic Limb in Mice With Type 2 Diabetes. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 10(19), e023491.

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Isolation and Sorting of Interstitial Cells

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For qPCR validations, interstitial cells were isolated as described above, from the whole ventricles of control mice or scar area and distal area in injured mice. In experiments with Coll1a1^eGFP mice, GFP⁺DRAQ5⁺PI^- cells were sorted on Aria II with 130 μm nozzle. In absence of a fluorescent reporter (129, B6J mice), cells were first resuspended in blocking solution containing anti-mouse CD16/CD32 (1:600, Leinco Technologies, C381, clone 2.4G2) in 2% FCS 5min at RT, then stained with APC/Cyanine7 anti-mouse CD45 (1:660, BioLegend, 103115, clone 30-F11), PE-Cy7 anti-mouse CD31 (1:800, BD Biosciences, 561410, clone 390) on ice for 15min. After washing in PBS, cells were resuspended in 2%FCS+2mM EDTA and DAPI (50ng/ml) before sorting on the AriaII for DAPI^-CD45^-CD31^-cells.

Forte E., Skelly D.A., Chen M., Daigle S., Morelli K.A., Hon O., Philip V.M., Costa M.W., Rosenthal N.A, & Furtado M.B. (2020). Dynamic Interstitial Cell Response during Myocardial Infarction Predicts Resilience to Rupture in Genetically Diverse Mice. Cell Reports, 30(9), 3149-3163.e6.

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Skin Macrophage Phenotyping in LL37-induced Rosacea

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The skin samples of the LL37-induced rosacea mice model were cut into small pieces and processed in digestion buffer (1 mg/ml collagenase) for 2 hours at 37°C. Cells from the skin samples were passed through 40 μm nylon mesh to obtain a single-cell suspension, then the single cells were resuspended in 30% Percoll solutions and centrifuged. Subsequently, the single-cell suspension was stained with Zombie Aqua (Biolegend, USA) for 20 minutes, blocked with Fc block for 10 minutes (anti-mouse CD16/32 mAbs; Biolegend, USA), and finally stained with the fluorochrome-conjugated antibodies for 20 minutes. Cells were then washed and measured on CytoFLEX (Beckman Coulter, USA) flow cytometer and analyzed by CytExpert (Beckman Coulter, USA) software. M1 macrophages were defined as CD45⁺CD11b⁺F4/80⁺CD86⁺cells. M2 macrophages were defined as CD45⁺CD11b⁺F4/80⁺CD206⁺ cells.
The following antibodies from BioLegend (CA, USA) were used: FITC anti-mouse/human CD11b (M1/70), APC/Cyanine7 anti-mouse CD45 (S18009F), APC anti-mouse F4/80 (BM8), PE/Cyanine7 anti-mouse CD206 (C068C2), Brilliant Violet 421 anti-mouse CD86 (GL-1).

Tang S., Hu H., Li M., Zhang K., Wu Q., Liu X., Wu L., Yu B, & Chen X. (2024). OPN promotes pro-inflammatory cytokine expression via ERK/JNK pathway and M1 macrophage polarization in Rosacea. Frontiers in Immunology, 14, 1285951.

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Comprehensive Immune Profiling of Tumors

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Tumors were enzymatically digested to single cell suspensions and filtered twice through 70 μm filters. Cells were then resuspended in staining buffer (DPBS containing 3% FBS) and stained with the indicated antibodies: APC/Cyanine7 anti-mouse CD45 (Cat. 147717, BioLegend), PE anti-mouse CD3 (Cat. 100205, BioLegend), APC anti-mouse CD8a (Cat. 100711, BioLegend), PerCP/Cyanine5.5 anti-human/mouse Granzyme B (Cat. 372211, BioLegend), VioletFluor™ 450 Anti-Mouse IFN gamma (Cat 75-7311-U025, Tonbo Bioscience), Brilliant Violet 510™ anti-mouse/human CD11b (Cat. 101245, BioLegend), PE anti-mouse Ly-6C (Cat. 128007, BioLegend), FITC anti-mouse Ly-6G (Cat.127605, BioLegend), PerCP/Cyanine5.5 anti-mouse F4/80 (Cat. 123127, BioLegend). Flow cytometry was performed on BD FACS Symphony machines. Flow analyses were performed using FlowJo 10.6.1 software (TreeStar). All flow antibodies were used at a 1:200 dilution.

Chen W., Liao Y., Sun P., Tu J., Zou Y., Fang J., Chen Z., Li H., Chen J., Peng Y., Wen L, & Xie X. (2024). Construction of an ER stress-related prognostic signature for predicting prognosis and screening the effective anti-tumor drug in osteosarcoma. Journal of Translational Medicine, 22, 66.

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Comprehensive Erythroid Cell Analysis

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Whole blood was removed by retroorbital bleed and stained for flow cytometry (BD FACSCanto II) using the following antibodies or fluorescent dyes: Biotin anti-mouse CD71 REAfinity (130-109-572, Miltenyi), anti-biotin-PE (130-113-291, Miltenyi), 0.0025 µg/µL PE-Cyanine7 Anti-Mouse TER-119/Erythroid Cells (116222, BioLegend), 0.0025 µg/µL APC-Cyanine7 Anti-Mouse CD45 (103116, BioLegend), 100 nM Thiazol orange (390062, Sigma Aldrich), 500 nM MitoTracker Deep Red FM (8778, Cell Signaling), and 2.5 µg/mL 7-AAD live-dead viability staining (420404, BioLegend).

Alula K.M., Delgado-Deida Y., Callahan R., Till A., Underwood L., Thompson W.E., Souza R.F., Dassopoulos T., Onyiah J., Venuprasad K, & Theiss A.L. (2023). Inner mitochondrial membrane protein Prohibitin 1 mediates Nix-induced, Parkin-independent mitophagy. Scientific Reports, 13, 18.

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Quantification of Autoreactive PMEL T Cells

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Skin infiltration of autoreactive PMEL CD8⁺ T cells in the footpad was quantified by flow cytometry (Cytex Aurora cytometer). Footpad skin was harvested at week 4 after vitiligo induction and treated with RPMI-1640 media (Gibco, #11875093) containing 2.5 mg/mL of collagenase IV (Sigma-Aldrich, #C6885) and 1 mg/mL deoxyribonuclease I (Sigma-Aldrich, #DN25) at 37 °C for 45 min. Skin samples were manually crushed through a 100-μm cell strainer (Fisher Scientific, #22363549). The following dye and flow cytometry antibodies were used for staining CD8⁺ and Thy1.1⁺ double-positive PMEL T cells that originated from the donor mice: TruStain FcX PLUS (anti-mouse CD16/32, Biolegend, #156604), Zombie Aqua™ Fixable Viability Kit (Biolegend, #423101), APC-Cyanine7 anti-mouse CD45 (Biolegend, #103116), PerCP-Cyanine5.5 anti-mouse CD8b (Biolegend, #126610), and FITC anti-mouse CD90.1 (Thy1.1, Biolegend, #202503). Cells were fixed by FluoroFix™ fixation buffer (Biolegend, #422101) prior to flow cytometry analysis.

Tang Q., Fakih H.H., Zain UI Abideen M., Hildebrand S.R., Afshari K., Gross K.Y., Sousa J., Maebius A.S., Bartholdy C., Søgaard P.P., Jackerott M., Hariharan V., Summers A., Fan X., Okamura K., Monopoli K.R., Cooper D.A., Echeverria D., Bramato B., McHugh N., Furgal R.C., Dresser K., Winter S.J., Biscans A., Chuprin J., Haddadi N.S., Sherman S., Yıldız-Altay Ü., Rashighi M., Richmond J.M., Bouix-Peter C., Blanchard C., Clauss A., Alterman J.F., Khvorova A, & Harris J.E. (2023). Rational design of a JAK1-selective siRNA inhibitor for the modulation of autoimmunity in the skin. Nature Communications, 14, 7099.

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