Apc conjugated anti mouse cd45

Manufactured by BioLegend

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APC-conjugated anti-mouse CD45 is a monoclonal antibody that binds to the mouse CD45 surface antigen. CD45 is a transmembrane protein tyrosine phosphatase that is expressed on the surface of all leukocytes.

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Anti-CD45 (228 citations) CD45-APC (97 citations) Anti-CD45-APC (42 citations) Anti-mouse CD45 (39 citations) APC anti-mouse CD45 (32 citations) APC-conjugated anti-CD45 (7 citations) APC conjugated anti-mouse CD45 antibody (4 citations) APC-conjugated CD45 (3 citations) APC/Cy7-conjugated anti-mouse CD45 antibody (3 citations) APC/Cy7-conjugated rat anti-mouse CD45 antibody (2 citations)

9 protocols using apc conjugated anti mouse cd45

Analyses of Inflammatory Responses in Mice

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4-EG, triphenyltetrazolium chloride (TTC), Alexa Fluor 594 goat-anti rabbit IgG secondary antibody, Evans blue, Ficoll PM 400, and LPS (Escherichia coli O55:B5) were purchased from MilliporeSigma (St. Louis, MO, USA). Zinc protoporphyrin (ZnPP) was purchased from Alfa Aesar (Haverhill, MA, USA). 7-aminoactinomycin D (7-AAD), Alexa Fluor 488-conjugated anti-mouse CD45 (clone: 30-F11), APC-conjugated anti-mouse CD45 (clone: 30-F11), PE/Cy7-conjugated anti-mouse CD11b (clone: M1/70), PE-conjugated anti-mouse CD11b (clone: M1/70), PE/Cy7-conjugated anti-mouse CD86 (clone: GL-1), PE/Cy7-conjugated anti-mouse CD68 (clone: FA-11), Alexa Fluor 647-conjugated anti-mouse CD31 (clone: MEC13.3), PE/Cy7-conjugated anti-mouse ICAM-1 (clone: HA58), and PE/Cy7-conjugated anti-mouse VCAM-1 (clone: 429, MVCAM.A) antibodies for flow cytometer analysis were purchased from BioLegend (San Diego, CA, USA). PE-conjugated anti-mouse E-selectin (clone: 10E9.6) antibody for flow cytometry analysis was purchased from BD Biosciences (San Diego, CA, USA). Alexa Fluor 488 anti-mouse Iba1 (EPR16588) antibody for immunohistochemistry (IHC) was purchased from Abcam (Cambridge, MA, USA). Anti-mouse HO-1 (10701-1-AP) antibody for flow cytometer and IHC was purchased from Proteintech (Chicago, IL, USA).

Weng W.T., Kuo P.C., Scofield B.A., Paraiso H.C., Brown D.A., Yu I.C, & Yen J.H. (2022). 4-Ethylguaiacol Modulates Neuroinflammation and Promotes Heme Oxygenase-1 Expression to Ameliorate Brain Injury in Ischemic Stroke. Frontiers in Immunology, 13, 887000.

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Isolation and Characterization of Murine Splenocytes

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Mouse splenocytes were collected aseptically from spleens of mice by mincing the spleen tissues in a sterile Petri plate, and the erythrocytes were lysed in lysis buffer (10 mmol/L KHCO₃, 150 mmol/L NH₄Cl, 10 mmol/L ethylenediaminetetraacetic acid, pH 7.4). Splenocytes were prepared from tumor-grafted mice or naïve mice. ACK (Ammonium–Chloride–Potassium) Lysing buffer was used to lyse erythrocytes. Splenocytes were stained using APC-conjugated anti-mouse CD3e antibody, APC-conjugated anti-mouse CD4, FITC-conjugated anti-mouse CD8a, APC/Cy7-conjugated anti-mouse CD11b, APC-conjugated anti-mouse CD45, and PE-conjugated anti-mouse Ly6G/Ly6c (Gr-1) (1:200, BioLegend, San Diego, CA, USA) following the manufacturer’s instructions. Living cells were assessed by using 1 mg/mL Propidium iodide (PI). Analyses of fluorescence staining were performed with a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA). Data were collected and analyzed using Kaluza^® for Gallios-Acquisition and Flow Analysis Software (Beckman Coulter). We collected at least 10,000 viable cell events per sample in each experiment.

Ma L., Sakamoto Y., Kanai A., Otsuka H., Takahashi A., Kakimi K., Imai T, & Shimokawa T. (2020). Characterization of a Novel Murine Colon Carcinoma Subline with High-Metastatic Activity Established by In Vivo Selection Method. International Journal of Molecular Sciences, 21(8), 2829.

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Mouse Lymphocyte Isolation and Phenotyping

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Mouse spleens were dissected out, ground, and filtered, and the filtrate was slowly added into 6 mL of mouse lymphocyte separation solution (Beijing Dakewe Biotechnology Co. Ltd., Beijing, China) along the wall of the centrifuge tube to isolate the mouse lymphocytes. The antibody system was configured with 100 μL of FACE solution (saline containing 2% fetal bovine serum) and 1 μL each of Brilliant Violet 510™-conjugated anti-mouse CD3, FITC-conjugated anti-mouse CD4, PerCP/Cyanine5.5-conjugated anti-mouse CD8a, APC-conjugated anti-mouse CD45 and PE-conjugated anti-mouse CD69 antibodies (BioLegend, San Diego, CA, USA) to every sample tube. After mixing, the cells were incubated at 4°C for 30 min in the dark. The excess antibodies were washed off with FACE solution, and the supernatant was discarded after centrifugation at 8000 rpm for 1 min. The sediment was dissolved with 200 μL of 4% paraformaldehyde in the dark for 20 min and the cells were detected by flow cytometry.

Yang H., Lei G., Deng Z., Sun F., Tian Y., Cheng J., Yu H., Li C., Bai C., Zhang S., An G, & Yang P. (2024). An Engineered Influenza a Virus Expressing the Co-Stimulator OX40L as an Oncolytic Agent Against Hepatocellular Carcinoma. Journal of Hepatocellular Carcinoma, 11, 1-13.

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Isolation and Analysis of Retinal Endothelial Cells

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Retinas were dissected from freshly collected eyes of P6 neonates and digested with LiberaseTM (Sigma-Aldrich, CAT#5401127001, 0.26 U/mL) and deoxyribonuclease I (Sigma-Aldrich, CAT#D4527, 10 mg/mL) in PBS (1 mL per one retina) at PBS at 37°C for 30 min under constant rotation. At 15 min of digestion period, the suspension was triturated 10 times through a 18G needle to dissociate the clumps. The reaction was stopped by adding BSA (final 1%) and EDTA (final 0.5 mM) and centrifuged at 400 × g at 4°C for 10 min. The pellet was further washed once with FACS buffer (0.5% BSA/0.5 mM EDTA in PBS) and stained with phycoerythrin-conjugated anti-mouse CD31 (1:200, BioLegend, CAT#102508), allophycocyanin (APC)-conjugated anti-mouse CD45 (1:300, BioLegend, CAT#103111) and APC-conjugated TER119 (1:300, BioLegend, CAT#116212) antibodies for 1 hour on ice. The retinal cells were washed twice and treated with DAPI to exclude dead cells. CD31+/CD45-/TER119- cells were sorted using BD FACSAria™ II (BD Biosciences). The cells from 4–6 pooled retinas were sorted in either buffer RLT from RNeasy Micro Kit (Qiagen, CAT#74004) supplemented with β-mercaptoethanol (Sigma-Aldrich, CAT#M3148) or 0.1% BSA/PBS for RNA-seq or ATAC-seq, respectively.
Individual biological replicates for RNA-seq or ATAC-seq represent pooled sorted cells from 4–6 genotype-matched pups (one retina was used for FACS per pup).

Yanagida K., Engelbrecht E., Niaudet C., Jung B., Gaengel K., Holton K., Swendeman S., Liu C.H., Levesque M.V., Kuo A., Fu Z., Smith L.E., Betsholtz C, & Hla T. (2020). Sphingosine 1-phosphate receptor signaling establishes AP-1 gradients to allow for retinal endothelial cell specialization. Developmental cell, 52(6), 779-793.e7.

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Molecular Mechanisms Underlying mTOR Signaling Regulation

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The following antibodies were purchased from Cell Signaling Technology: anti-phospho- IRS1 (S1101 Cat # 2385), anti-phospho-S6 (S240/244, Cat # 5364), anti-phospho-TSC2 (S939 Cat # 3615), anti-TSC2 (Cat # 3990), anti-PTEN (Cat # 138G6), anti-Phospho-NF-kB p65 (S536 Cat # 93H1), anti-NF-kB p65 (Cat # D14E12), anti-phospho-4EBP1 (S65 Cat # 13443), anti-4EBP1(Cat # 9644), and anti-phospho-GSK3β (S9, Cat # 5558). Anti-IRS1 (Cat # 06–248) antibody was purchased from Merck Millipore. Anti-DDIT4 (Cat#10638–1-AP) and anti-SESN2 (Cat # 10795–1-AP) antibodies were purchased from ProteinTech Group. HRP conjugated anti-β-actin (Cat # A3854) was purchased from Sigma. HRP-linked mouse IgG (Cat # NA931V) and rabbit IgG (Cat # NAV934V) were purchased from GE Healthcare Life Sciences. IgG (Isotype control Cat # 400107), FITC conjugated anti-human CD45 (Cat # 304006), and APC conjugated anti-mouse CD45 (Cat # 103112) were purchased from BioLegend. Anti-S6 (Cat # SC74459) antibody was purchased from Santa Cruz laboratories. LGB-321 (Cat # A14420), AZD1208 (Cat # A13203), and IKK16 (# A12836) were purchased from Adooq bioscience. TNF-α (Cat # PHC3015) was purchased from Fisher scientific.
Supplementary Methods include a description of the Western Blot, RNA-Seq data analysis, GSEA analysis, RPPA, Active module analysis, Lentiviral production, the NF-κB reporter assay, and Statistics.

Lim J.T., Singh N., Leuvano L.A., Calvert V.S., Petricoin E.F., Teachey D.T., Lock R.B., Padi M., Kraft A.S, & Padi S.K. (2020). PIM kinase inhibitors block the growth of primary T-cell acute lymphoblastic leukemia: Resistance pathways identified by network modeling analysis. Molecular cancer therapeutics, 19(9), 1809-1821.

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Quantification of CD123 Expression

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CD123 expression was detected using a PE-conjugated anti-human CD123 antibody (Clone 6H6, Catalog No. 306006, BioLegend; San Diego, CA, USA). Samples were analyzed on a NovoCyte 3000 Flow Cytometer. CD123 cell surface expression was quantitated with the use of BD Quantibrite PE Phycoreythrin Fluorescence Quantitation kit (Catalog No. 340495, BD Biosciences, San Jose, CA, USA) following the manufacturer’s protocol.
Peripheral blood was stained with APC-conjugated anti-mouse CD45 (BioLegend Catalog No. 103112), Pacific blue conjugated anti-human CD45 (BioLegend Catalog No. 304029) and FITC conjugated anti-human CD3 (BioLegend Catalog No. 317306) antibodies following incubation with human BD Fc block (BD Biosciences, Catalog No. 564219) to prevent non-specific antibody binding.

Barwe S.P., Kisielewski A., Bonvini E., Muth J., Davidson-Moncada J., Kolb E.A, & Gopalakrishnapillai A. (2022). Efficacy of Flotetuzumab in Combination with Cytarabine in Patient-Derived Xenograft Models of Pediatric Acute Myeloid Leukemia. Journal of Clinical Medicine, 11(5), 1333.

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Comprehensive Flow Cytometry Profiling of CSCs and BMSCs

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Flow cytometry analysis was conducted to evaluate the cell surface marker expressions of the CSCs. CSCs with various treatments were harvested and trypsinized into a single cell suspension. Subsequently, cells were incubated with fluorochrome-conjugated antibodies, including FITC-conjugated anti-human cluster of differentiation (CD) 24 (eBioscience, CA, USA), APC-conjugated anti-human CD133 (Biolegend, CA, USA) at 4 °C in the dark. The appropriated isotype-matched antibodies were used as negative controls. After 30-min incubation, the samples were centrifuged and washed with PBS for flow cytometry analysis. Similarly, the cell surface marker expressions of the BMSCs were also evaluated by flow cytometry. The conjugated-specific antibodies used were as follows: PE-conjugated anti-mouse CD14 (Biolegend, CA, USA), APC-conjugated anti-mouse CD34 (Biolegend, CA, USA), APC-conjugated anti-mouse CD45 (Biolegend, CA, USA), PE-conjugated anti-mouse CD44 (Biolegend, CA, USA), PE-conjugated anti-mouse CD73 (Biolegend, CA, USA), APC-conjugated anti-mouse CD105 (Biolegend, CA, USA), FITC-conjugated anti-mouse CD166 (abcam, CA, USA), FITC-conjugated anti-mouse CD29 (Biolegend, CA, USA). Cell analysis was performed using a Flow cytometer (BD Biosciences, CA, USA) and FolwJo software version 7.6 (FlowJo LLC, OR, USA).

Jingjing H., Hongna H., Xiaojiao W., Yan G., Yuexue Z, & Yueqiang H. (2021). Bie Jia Jian pill enhances the amelioration of bone mesenchymal stem cells on hepatocellular carcinoma progression. Journal of Natural Medicines, 76(1), 49-58.

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Comprehensive Molecular and Cellular Analysis Protocol

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Flow cytometry antibodies and reagents: FITC-conjugated anti-mouse CD11b, APC-conjugated anti-mouse CD45, PE-conjugated anti-mouse LAMP1 and PE-conjugated anti-mouse CD86 were purchased from Biolegend (USA), anti-CD16/CD32 Fc Block and myelin removal reads were purchased from Miltenyi Biotec (Germany). Western blot and immunofluorescence (IF) antibodies: IBA-1 (R&D systems, USA); V GLUT1 (Santa Cruz Biotechnology, USA); LAMP1 and CD68 were purchased from Abcam (United Kingdom); albumin, PSD95 and PKM2 were purchased from Proteintech (China). β-catenin, Cyclin-D1, c-Myc, ENO1, PFKFB3, HK1, LDHA, PDK1, GLUT1 were purchased from Cell Signaling Technology (USA), horseradish peroxidase (HRP)-linked goat anti-rabbit IgG (Fcmacs, China), NeuN (Santa Cruz Biotechnology), donkey anti-rat IgG H&L (Alexa Fluor® 647) (Abcam), goat anti-mouse IgG-TRITC (Abcam), rabbit anti-goat IgG-FITC (Fcmacs). ELISA kit: Maltose and Glucose assay kit (RayBiotech, USA), l-lactate assay kit (Cayman, USA), interleukin (IL)-6 (Fcmacs, China), IL-1β (Fcmacs, China). TRIzol reagent and SYBR green dye were bought from Invitrogen (Carlsbad, USA). PKM2 overexpression plasmid was purchased from Nanjing Jereh Company (China), and RFectPM Eukaryotic DNA Transfection Kit was purchased from Changzhou EMI Company (China). β-catenin protein inhibitor (KYA1797K) was purchased from MCE Company (USA).

Lu L., Wang H., Liu X., Tan L., Qiao X., Ni J., Sun Y., Liang J., Hou Y, & Dou H. (2021). Pyruvate kinase isoform M2 impairs cognition in systemic lupus erythematosus by promoting microglial synaptic pruning via the β-catenin signaling pathway. Journal of Neuroinflammation, 18, 229.

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Retinal Cell Isolation and Analysis

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Retinas were dissected from freshly collected eyes and digested with Liberase^TM (Sigma‐Aldrich, CAT#5401127001, 0.26 U/ml) and deoxyribonuclease I (Sigma‐Aldrich, CAT#D4527, 10 mg/ml) in PBS (1 ml per one retina) at PBS at 37°C for 30 min. The reaction was stopped by adding SVF (final 1%) and centrifuged at 400 × g at 4°C for 10 min. The pellet was further washed once with FACS buffer (0.5% BSA/0.5 mM EDTA in PBS) and stained with APC‐conjugated anti‐mouse CD45 (1:300, BioLegend, CAT#103112), BV510‐conjugated anti‐mouse CD11b (1:300, BioLegend, #CAT101263), and PE‐conjugated anti‐mouse Ly6G (1:300, BD Pharmingen, #CAT5551461) antibodies for 1 h on ice. The retinal cells were washed twice and treated with DAPI to exclude dead cells. CD31⁺/CD45⁻/TER119⁻ cells were analyzed using BD FACSAria™ II (BD Biosciences).

Niaudet C., Jung B., Kuo A., Swendeman S., Bull E., Seno T., Crocker R., Fu Z., Smith L.E, & Hla T. (2023). Therapeutic activation of endothelial sphingosine‐1‐phosphate receptor 1 by chaperone‐bound S1P suppresses proliferative retinal neovascularization. EMBO Molecular Medicine, 15(5), e16645.

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