Sdf 1

Manufactured by R&D Systems

Sourced in United States, United Kingdom

SDF-1 is a recombinant human protein that functions as a chemoattractant for a variety of cell types, including hematopoietic stem and progenitor cells. It is a member of the CXC chemokine family and binds to the CXCR4 receptor.

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Mouse CXCL12/SDF-1 alpha Quantikine ELISA Kit (14 citations) Human CXCL12/SDF-1 alpha Quantikine ELISA Kit (8 citations) CXCL12/SDF-1 (7 citations) Human CXCL12/SDF-1 ELISA Kit (4 citations) Recombinant human CXCL12/SDF-1 (4 citations) Human CXCL12/SDF-1 DuoSet ELISA (3 citations) SDF‐1 ELISA kit (3 citations) Quantikine Mouse VEGF- and SDF-1 Immunoassays (2 citations) Recombinant mouse SDF-1 (2 citations) Human CXCL12/SDF-1 kit (2 citations)

65 protocols using sdf 1

SDF-1 and Wnt Signaling Modulation

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Once the cells had attached, the medium was changed to DMEM without serum. After 24 h, the cells were divided into four groups and treated as follows: SDF-1 group, 100 ng/ml SDF-1 (cat. no. ab79959; Abcam); SDF-1 + AMD3100 group, 100 ng/ml SDF-1 and 5 µg/ml AMD3100 (Sigma-Aldrich; Merck KGaA); SDF-1 + DKK-1 group, 100 ng/ml SDF-1 and 200 ng/ml DKK-1 (R&D Systems, Inc., Minneapolis, MN, USA); and control group, PBS at a dose equivalent to the SDF-1, AMD3100 and DKK-1 in the other groups.
Following incubation at 37°C for 24 h, the cellular total protein and nuclear protein were extracted for western blotting (WB) and the total RNA was extracted for reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

Tang H., Xu Y., Zhang Z., Zeng S., Dong W., Jiao W, & Hu X. (2019). SDF-1/CXCR4 induces epithelial-mesenchymal transition through activation of the Wnt/β-catenin signaling pathway in rat chronic allograft nephropathy. Molecular Medicine Reports, 19(5), 3696-3706.

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Intracellular Calcium Imaging of DRG Neurons

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The response of acutely cultured DRG neurons to the chemokines SDF-1 and MCP-1 (500 ng/ml R&D Systems) were recorded using intracellular calcium imagining following a standard protocol previously used in our laboratory [20 (link)]. Briefly, acutely cultured DRG cells were loaded with fura-2 AM (3uM, Invitrogen, Carlsbad, CA) for 25 minutes at room temperature in a balanced salt solution (BSS) [NaCl (140 mM), Hepes (10 mM), CaCl2 (2 mM), MgCl2 (1 mM), Glucose (10 mM), KCl (5 mM)]. The cells were rinsed with the BSS and mounted onto a chamber that was placed onto the inverted microscope and continuously perfused with BSS at a rate of 2 ml/min. Intracellular calcium ([Ca2+]i) was measured by digital video microfluorometry with an intensified CCD camera coupled to a microscope and MetaFluor software. Cells were illuminated with a 150W xenon arc lamp, and the excitation wavelengths of the fura-2 (340/380 nm) were selected by a filter changer. Chemokines were applied for two minutes directly into the coverslip bathing solution after the perfusion was stopped. If no response was seen within 1 minute, the chemokine was washed out. For all experiments, SDF-1 (500 ng/ml, R&D Systems) and MCP-1 (500 ng/ml, R&D Systems), capsaicin (100 nM), high K + (50 mM) and ATP (100 μM) were added to the cells.

Menichella D.M., Abdelhak B., Ren D., Shum A., Frietag C, & Miller R.J. (2014). CXCR4 chemokine receptor signaling mediates pain in diabetic neuropathy. Molecular Pain, 10, 42.

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Stimulation of HL-60 and Mouse Lineage Negative Cells with SDF-1

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HL-60 cells (ATCC CCL-240) were obtained from the American Type Culture Collection (Manassas, VA) and maintained in Iscove’s Modified Dulbecco’s Medium (IMDM) with 20% FBS. HL-60 cells were incubated in IMDM +20% FBS with and without 50 ng/ml SDF-1 (R&D, Minneapolis, MN) for two and 24 hours, respectively. This concentration of SDF-1 has been shown to elicit optimal responses in numerous of our assays [12 (link),18 (link),19 (link),21 (link),41 (link)]. C57Bl/6 strain mice were used to isolate lineage negative bone marrow cells. The Indiana University Committee on Use and Care of Animals approved the mouse studies. Mouse lineage negative cells were isolated using the Miltenyi Biotech (San Diego, CA) mouse Lineage Cell Depletion Kit. After lineage depletion, lineage negative cells were incubated in IMDM +10% FBS and stimulated with or without 50 ng/ml SDF-1 (R&D, Minneapolis, MN) for two and 24 hours, respectively.

Messina-Graham S, & Broxmeyer H. (2016). SDF-1/CXCL12 Modulates Mitochondrial Respiration of Immature Blood Cells in a Bi-phasic Manner. Blood cells, molecules & diseases, 58, 13-18.

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Transwell Assay for HSPC Migration

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In vitro migration of HSPCs was performed using Transwells (6.5mm diameter inserts; 5μm pore size; Corning). FACS-purified HSPCs were loaded onto Transwell inserts (10⁵ cells/well in 100μl of medium). The lower chambers contained 600μl of RPMI medium supplemented with 10% FCS and with SDF-1 (100ng/ml) or without SDF-1 (R&D systems). Cells were incubated for 4 hours at 37°C and 5%CO₂. Migrating cells from the lower chambers were collected and counted by FACS with normalization using Countbright Absolute counting beads (Invitrogen). The percentage of migrated cells was calculated by dividing the absolute number of migrated cells by the input cell number.

Rao T.N., Marks-Bluth J., Sullivan J., Gupta M.K., Chandrakanthan V., Fitch S.R., Ottersbach K., Jang Y.C., Piao X., Kulkarni R.N., Serwold T., Pimanda J.E, & Wagers A.J. (2015). High-level Gpr56 expression is dispensable for the maintenance and function of hematopoietic stem and progenitor cells in mice. Stem cell research, 14(3), 307-322.

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Assessing CXCR4 and CXCR7 Roles in Melanoma Osteotropism

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Further experiments assessed the contribute of either CXCR4 or CXCR7 in conditioning the osteotropism of melanoma cells. Therefore, their expression was restrained by small interfering RNAs (siRNAs) using the following primers: 5′-GGCAGUCCAUGUCAUCUACTT-3′ and 5′-GUAGAUGACAUGGACUGCCTT-3′ for CXCR4; 5′-GGAUGACACUAAUUGUUAGTT-3′ and 5′-CUAACAAUUAGUGUCAUCCTT-3′ for CXCR7 (Life Technologies, CA, USA). Briefly, 1 × 10⁶ LCP and SK-Mel28 cells were treated for 48 h with 3.75 µl/ml of Lipofectamine 3000 Reagent (Invitrogen, CA, USA) to induce transient transfection in presence of anti-CXCR4 (30 nmol/l) or anti-CXCR7 (60 nmol/l) siRNAs. Cells treated with 3.75 µl/ml of Lipofectamine 3000 Reagent or scramble probes (Ambion) were the controls.
Since both CXCR4 and CXCR7 share the SDF-1 ligand, silenced melanoma cells were explored in their propensity to migrate and invade toward human-recombinant SDF-1 (R&D Systems, MN, USA) either in presence or absence of Exos. The optimal concentration of SDF-1 used in these experiments, namely 100 ng/ml, was similar to that detected in BCM by ELISA (96.4 ± 17.2 ng/ml).

Mannavola F., Tucci M., Felici C., Passarelli A., D’Oronzo S, & Silvestris F. (2019). Tumor-derived exosomes promote the in vitro osteotropism of melanoma cells by activating the SDF-1/CXCR4/CXCR7 axis. Journal of Translational Medicine, 17, 230.

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Iso, SDF-1, and AMD3100 Protocol for H9C2 Cells

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All chemicals and bioagents used in the present study, including Iso, AMD3100, Gi inhibitor Pertussis toxin (all from Sigma-Aldrich; Merck KGaA), SDF-1 (R&D Systems, Inc.) were purchased from commercial sources and authenticated by the providers. Their concentrations, Iso (100 µM), SDF-1 (500 ng/ml), and AMD3100 (100 ng/ml), used on H9C2 cells were based on pilot studies by the authors and consistent with a similar study previously reported (39 (link)).

Cheng M., Chen C., Yu K., Lv X., Zeng Q., Dong N, & Zhu F. (2022). Ablation of CXCR4 expression in cardiomyocytes exacerbates isoproterenol-induced cell death and heart failure. International Journal of Molecular Medicine, 51(2), 13.

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Quantifying MSC Migration via Fluorescence

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A migration assay was performed using FluoroBlok insert (BD Biosciences) containing 5 × 10⁴ MSCs placed above 1.0 × 10⁶ myoblasts seeded on cell culture plates. Control cultures comprised inserts placed over cell culture plates without cells supplemented with 150 ng/ml SDF-1 (R&D Systems). The FluoroBlok insert system allows the quantification of cells that have migrated through the insert by a bottom-reading fluorometer. After culturing for 6 h, MSCs were stained with 4 μg/ml Calcein-AM for 30 min at 37 °C, and fluorescence signals of the migrated cells were measured using a SH-9000 plate reader (COLONA ELECTRIC, Ibaraki, Japan).

Iseoka H., Miyagawa S., Saito A., Harada A, & Sawa Y. (2020). Role and therapeutic effects of skeletal muscle-derived non-myogenic cells in a rat myocardial infarction model. Stem Cell Research & Therapy, 11, 69.

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iPSC-MSC Adhesion Assays under Flow

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For adhesion assays, 10⁵ iPSC-MSCs were allowed to rest for 3 min on a laminar flow chamber slide (μ-slide, ibiTreat; IBIDI Systems, Munich, Germany) mounted on an inverted microscope as previously described [13 (link)]. Briefly, flow chambers were precoated with 2 μg/mL VCAM-1 fusion protein and cocoated with SDF-1, both from R&D Systems (1 μg/mL). Subsequently, HBSS/0.1% BSA (prewarmed to 37°C) was flushed through the chambers at the indicated calculated shear stresses with increases in steps between 0.35 and 15 dyn/cm² every 30 s. Images were taken, and the adherent cells were counted in four fields for every condition.

Moslem M., Eggenschwiler R., Wichmann C., Buhmann R., Cantz T, & Henschler R. (2017). Kindlin-2 Modulates the Survival, Differentiation, and Migration of Induced Pluripotent Cell-Derived Mesenchymal Stromal Cells. Stem Cells International, 2017, 7316354.

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Quantifying SDF1-induced cAMP in SJCRH30 cells

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SJCRH30 cells were grown as confluent monolayers in a 96-well plate. Before stimulation, cells were incubated for 30 min with mAb and 1 mM IBMX (Sigma-Aldrich Japan, Tokyo, Japan). Cells were stimulated with 100 nM SDF1 (R&D Systems) diluted in HBSS/3-isobutyl-1-methylxanthine (IBMX) for 10 min, after which they were incubated for an additional 10 min with 25 μM forskolin (Sigma-Aldrich Japan) to stimulate cyclic AMP (cAMP) production. Then, cells were washed twice in ice-cold HBSS/IBMX and solubilized, and cAMP was assayed using the cAMP Parameter Assay Kit (R&D Systems).

Kashima K., Watanabe M., Sato Y., Hata J., Ishii N, & Aoki Y. (2014). Inhibition of metastasis of rhabdomyosarcoma by a novel neutralizing antibody to CXC chemokine receptor-4. Cancer Science, 105(10), 1343-1350.

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Osteogenic Differentiation Assay for AML

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AMD3100, ascorbic acid, β-glycerol phosphate, hydrocortisone, dexamethasone, and alizarin red were purchased from Sigma-Aldrich (St. Louis, MO). SDF-1 and BMP-2 were obtained from R&D Systems (Minneapolis, MN). A murine monoclonal antibody to CXCR4 conjugated to APC (R&D Systems) was used to assay cell-surface CXCR4 levels on AML cells, and APC-conjugated annexin-V (BD Biosciences, San Jose, CA) was used for flow cytometric assays of AML cell apoptosis.

Kremer K.N., Dudakovic A., McGee-Lawrence M.E., Philips R.L., Hess A.D., Smith B.D., van Wijnen A.J., Karp J.E., Kaufmann S.H., Westendorf J.J, & Hedin K.E. (2014). Osteoblasts Protect AML Cells from SDF-1-Induced Apoptosis. Journal of cellular biochemistry, 115(6), 1128-1137.

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