Alexa fluor 488

Manufactured by R&D Systems

Sourced in United States

Alexa Fluor 488 is a fluorescent dye that can be used for labeling proteins, cells, and other biomolecules. It has an excitation maximum at 488 nm and an emission maximum at 519 nm, making it compatible with common fluorescence detection equipment.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Murine anti human cd45 bv421 clone hi30 monoclonal antibody, by BD (2 mentions) Isotype control mouse antibodies for igg1 and igm, by R&D Systems (2 mentions) Anti human cd276 b7 h3 pe cy7 clone mih42, by BioLegend (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Facsvantage cytometer, by BD (1 mentions) Trypsin, by Thermo Fisher Scientific (1 mentions) T1300, by Solarbio (1 mentions) Fluorescein isothiocyanate (fitc), by BioLegend (1 mentions) Laminin, by Agilent Technologies (1 mentions) Alexa fluor 546, by Thermo Fisher Scientific (1 mentions) Eclipse e600 microscope, by Nikon (1 mentions) Moflo astrio, by Beckman Coulter (1 mentions) Cd34 apc, by BD (1 mentions) Anti sca 1 apc cy7, by BD (1 mentions) 7 aad, by BD (1 mentions) Flow cytometry staining buffer, by R&D Systems (1 mentions) Facscalibur flow cytometer, by BD (1 mentions) Percp efluor 710, by Thermo Fisher Scientific (1 mentions)

14 protocols using alexa fluor 488

Quantifying LDLR Expression in HepG2 Cells

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The LDLR expression level on the membrane surface of HepG2 cells was determined by flow cytometry. The cultured HepG2 cells were collected and washed with ice-cold flow cytometry staining buffer (R&D Systems, Minnesota, USA) three times. Cell pellets were resuspended in blocking IgG solution (1 μg IgG/10⁶ cells) for 15 min at 4°C to block nonspecific binding. Antibodies used include mouse anti-human LDLR monoclonal Alexa Fluor® 488 and mouse IgG1 isotype control Alexa Fluor® 488 (R&D Systems), while assays were performed according to the flow cytometry protocol for staining membrane-associated proteins (R&D Systems). The flow cytometric acquisition and data analysis were performed using a BD FACSCalibur flow cytometer and CellQuest software. Three independent flow cytometric experiments were performed.

Huang L., Cheng Y., Mu Y, & Li K. (2023). PCSK9-D374Y Suppresses Hepatocyte Migration through Downregulating Free Cholesterol Efflux Rate and Activity of Extracellular Signal-Regulated Kinase. Analytical Cellular Pathology (Amsterdam), 2023, 6985808.

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Bone Marrow Endothelial Cell Immunostaining

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Freshly isolated bone samples were cleaned thoroughly to remove the adherent muscles and then crushed in ice-cold PBS with a mortar and pestle. Whole bone marrow was digested with Collagenase at 37 °C for 20 min. After washing and filtering, single-cell suspensions were subjected to immunostaining. Cells were stained with Emcn (sc-65495, Santa Cruz, 1:100) antibody for 45 min. After washing in PBS, cells were stained with anti-rat APC (Jackson laboratories, 712-136-153, 1:50) and CD31-conjugated with Alexa Fluor 488 (R&Dsystems, FAB3628G, 1:50) for 45 min. After washing, data were acquired and analysed on BD FACSVantage cytometer (BD Biosciences).

Ramasamy S.K., Kusumbe A.P., Schiller M., Zeuschner D., Bixel M.G., Milia C., Gamrekelashvili J., Limbourg A., Medvinsky A., Santoro M.M., Limbourg F.P, & Adams R.H. (2016). Blood flow controls bone vascular function and osteogenesis. Nature Communications, 7, 13601.

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Endothelial Cell Barrier Regulation by Slit2

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The human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection. LPS (Escherichia coli O111:B4) and Slit2 ELISA kit (cat. no. HPA019511) were purchased from Sigma-Aldrich (Merck-KGaA), while aminophylline was purchased from Shandong Xinhua Pharmaceutical Co., Ltd. The Robo4 (cat. no. MAB50041), VE-cadherin (cat. no. AF1002), fibronectin (cat. no. AF1918) and integrin (cat. no. AF2045) primary antibodies were purchased from R&D Systems, Inc. Alexa Fluor 488- (cat. no. sc-362257) and 594- (cat. no. sc-362277) conjugated and GAPDH antibodies (cat. no. sc-47724) were purchased from Santa Cruz Biotechnology, Inc. The N-terminal Slit2 (Slit2-N) protein was purchased from Abcam. FBS, trypsin, penicillin/streptomycin and DMEM were purchased from Thermo Fisher Scientific, Inc. All other chemicals were purchased from Sigma-Aldrich (Merck KGaA).

Chen Q., Zhou X., Hou R., Zhou Z., Wang Z., Chen Y., Weng J, & Xu J. (2021). Aminophylline modulates the permeability of endothelial cells via the Slit2-Robo4 pathway in lipopolysaccharide-induced inflammation. Experimental and Therapeutic Medicine, 22(3), 1042.

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Visualizing MMP14 Recycling Dynamics

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Cells were transfected with mCherry-MMP14 and siNEDD9. Mouse monoclonal anti-MMP14 antibodies conjugated with AlexaFluor-488 (R&D Systems) were added to the cells for 1 hour at 37°C. Cells were washed with acidic strip solution (25 (link)), and the Ab/MMP14 complex was allowed to recycle for 1 hour at 37°C, followed by fixation with 4% PFA/PBS and processed for immunofluorescence (without permeabilization).

Loskutov Y.V., Kozyulina P.Y., Kozyreva V.K., Ice R.J., Jones B.C., Roston T.J., Smolkin M.B., Ivanov A.V., Wysolmerski R.B, & Pugacheva E.N. (2014). NEDD9/Arf6-Dependent Endocytic Trafficking of Matrix Metalloproteinase 14: A Novel Mechanism for Blocking Mesenchymal Cell Invasion and Metastasis of Breast Cancer. Oncogene, 34(28), 3662-3675.

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Isolation and Characterization of Rat BMSCs

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BMSCs were isolated from 6-week-old male Sprague-Dawley rat as described previously [35 (link)]. Care of the animals was in compliance with the Animal Ethics Committee of Shanxi Medical University. Briefly, the femora and tibiae were aseptically removed after euthanasia. The epiphyses were cut off and the marrow was flushed out using DMEM/F12 supplemented with 10% FBS (Gibco), 100 U/ml penicillin, 100 μg/ml streptomycin. After cultured for 2 days in an incubator (37°C, 5% CO₂), the medium was replaced to remove non-adherent cells. BMSCs were cultured to 90% confluence, trypsinized with 0.25% trypsin/0.53 mM ethylenediaminetetra acetic acid (EDTA) (T1300, Solarbio, Beijing, China) and passaged at 1:2.
The specific cell surface antigen markers of rat BMSCs were detected by flow cytometry (FCM, Beckman Coulter, Navios). The expression of antibodies for positive markers included CD44 (Alexa Fluor 488, R&D systems), CD90 (FITC, BioLegend) and CD29 (APC, MACS), and the negative markers included CD45 (PE-Vio770, MACS) and CD11b (PE, BioLegend).

Cao Y., Cheng P., Sang S., Xiang C., An Y., Wei X., Shen Z., Zhang Y, & Li P. (2021). Mesenchymal stem cells loaded on 3D-printed gradient poly(ε-caprolactone)/methacrylated alginate composite scaffolds for cartilage tissue engineering. Regenerative Biomaterials, 8(3), rbab019.

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Quantifying Satellite Cells in Muscle Tissue

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To assess the number of satellite cells in muscle tissue sections, immunostaining against transcription factor Pax7 (R&D Systems) was performed. laminin was used to delineate muscle fibers. Nuclei were labeled using DAPI. Nuclei located deep to the basal lamina and positive for Pax7 were counted as satellite cells. Satellite cells were counted in over 100 myofibers and reported as a ratio. Muscle sections (10 μm) were fixed with ice-cold acetone and methanol 60/40 (v/v) at – 20°C for 20 minutes, washed with PBS, incubated with a blocking solution (horse serum 1%) for 1 hour and then incubated with the primary antibody overnight at 4°C in a humidified chamber. The primary antibody Pax-7 (R&D Systems) and laminin (Dako, Glostrup, Denmark) excess was cleaned with PBS, incubated with their specific secondary antibodies, a goat anti-mouse Alexa Fluor® 488 (R&D Systems) and a goat anti-rabbit Alexa Fluor® 546 (Invitrogen Corporation, Carlsbad, CA, USA) for 1 hour, washed with PBS and then incubated with DAPI for 15 minutes. Slides were analyzed and images were captured using a Nikon Eclipse E600 microscope (Nikon Corporation, Tokyo, Japan). Immunofluorescences were performed in duplicate on two different muscle cryosections.

Thériault M.E., Paré M.È., Lemire B.B., Maltais F, & Debigaré R. (2014). Regenerative defect in vastus lateralis muscle of patients with chronic obstructive pulmonary disease. Respiratory Research, 15(1), 35.

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CXCR4 Expression-Based Cell Sorting

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Cell sorting was performed as previously described [9 (link)]. In brief, cells were blocked for 15 minutes at 4°C with agitation (0.5% FBS), incubated with human CXCR4 Alexa Fluor 488 or Alexa Fluor 700 monoclonal antibody (5 uL/1.0×10⁶ cells) (R&D Systems, Minneapolis, MN) for 30 minutes at 4°C with agitation, passed through a 0.40 μm sterile nylon mesh strainer and sorted into CXCR4^neg and CXCR4^high on a Beckman Coulter MoFlo Astrios.

Krook M.A., Hawkins A.G., Patel R.M., Lucas D.R., Van Noord R., Chugh R, & Lawlor E.R. (2016). A bivalent promoter contributes to stress-induced plasticity of CXCR4 in Ewing sarcoma. Oncotarget, 7(38), 61775-61788.

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Neuroblastoma Cell Detection Protocol

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Clinical-grade dinutuximab was generously provided by United Therapeutics (Silver Spring, MD, USA). Monoclonal anti-human neuroblastoma antibodies HSAN1.2 and 459 have been used extensively to detect neuroblastoma cells without binding in human bone marrow or blood [36 (link),37 (link),38 (link)]. Dinutuximab was directly conjugated to Alexa Fluor 647 and HSAN was directly conjugated to Alexa Fluor 488 by Pacific Immunology (Ramona, CA, USA). The 459 antibody was directly conjugated to PE-Cy7. Murine anti-human CD45 BV421 clone HI30 monoclonal antibody was purchased from BD Biosciences (San Jose, CA, USA). Human isotype control IgG1 directly conjugated with Alexa Fluor 647 (Dendritics, Lyon, France) and isotype control mouse antibodies for IgG1 and IgM directly conjugated with Alexa Fluor 488 were purchased from R&D Systems (Minneapolis, MN, USA). Anti-human CD276 (B7-H3) Pe-Cy7 clone MIH42 was purchased from Biolegend (San Diego, CA, USA).

Keyel M.E., Furr K.L., Kang M.H, & Reynolds C.P. (2023). A Multi-Color Flow Cytometric Assay for Quantifying Dinutuximab Binding to Neuroblastoma Cells in Tumor, Bone Marrow, and Blood. Journal of Clinical Medicine, 12(19), 6223.

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Quantifying Immune Cell EGFR Expression

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For mRNA expression of EGFR, cell subsets from C57Bl6 mice were collected at 24 h following 150 mg/kg 5-FU, 0.5 mg/kg IV EGF (R&D Systems, Minneapolis, MN), or 0.5 mg/kg subcutaneously G-CSF. mRNA EGFR expression analyses and analysis for other targets (CDK1, CDK2, CDK4, CDK6, p21, PUMA, and BCl-2) were performed according to manufacturer’s specifications (ThermoFisher Scientific, Waltham, MA). Relative expression of genes was calculated using delta-delta C_T analysis with normalization to GAPDH.
For protein expression of EGFR, cell subsets were isolated according to manufacturer’s specifications (Miltenyi Biotec, Auburn, CA). BM lineage-negative (lin⁻) cells were stained with CD34 APC (BD), anti-EGFR FITC (Abcam, Cambridge, MA), 7-AAD (BD), and anti-lineage antibody APC, anti-cKit PE, and anti-Sca-1 APC-Cy7 (BD, San Jose, CA). Phosphorylation of EGFR was performed using published methods [18 ].
G-CSFR expression was determined using ckit+Sca+Lin- (KSL) antibodies (BD) and anti-G-CSFR antibody Alexafluor 488 (R&D Systems).
For phospho-EGFR of human cells, cells were labeled with CD34+, and then cells were fixed and permeabilized as described [18 ]. Cells were labeled with anti-phospho-EGFR (Y1078) antibody (Cell Signaling, Danvers, MA). Isotype controls were included for all analyses.

Piryani S.O., Kam A.Y., Kliassov E.G., Chen B.J., Spector N.L., Chute J.P., Hsu D.S., Chao N.J, & Doan P.L. (2017). Epidermal growth factor and G-CSF signaling are synergistic for hematopoietic regeneration. Stem cells (Dayton, Ohio), 36(2), 252-264.

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Antibody Labeling and Carbohydrate Reagents

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Murine anti-DC-SIGN(R) (clone 120612), murine anti-LSECtin (SOTO-1) and goat anti-mouse IgG conjugated with Alexa Fluor 488 were purchased from R&D Systems, Santa Cruz Biotech, and Molecular Probes, respectively. Murine anti-feline CD2 (clone SKR2) was reported previously [17 (link)]. D1-4G2-4-15 (against flavivirus group antigen, [18 (link)]) hybridoma, yeast mannan, and N-acetylglucosamine β1-2 mannose (GlcNAcβ1-2Man disaccharide) were purchased from the American Type Culture Collection (ATCC), Sigma, and Dextra Laboratories, respectively.

Shimojima M., Takenouchi A., Shimoda H., Kimura N, & Maeda K. (2014). Distinct usage of three C-type lectins by Japanese encephalitis virus: DC-SIGN, DC-SIGNR, and LSECtin. Archives of Virology, 159(8), 2023-2031.

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