Mab310

Manufactured by R&D Systems

MAB310 is a monoclonal antibody product manufactured by R&D Systems. It recognizes and binds to an undisclosed target protein.

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Lab products found in correlation

8 protocols using mab310

Chemokine Separation and Co-Immunoprecipitation

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Chemokine mixtures or the OHD_4–12 were electrophoretically separated in non-denaturing or denaturing conditions using 16.5% polyacrylamide gel and then transferred to 0.2 μm nitrocellulose membrane using a semi-transfer apparatus (Biorad). Membranes were blocked in Tris-buffered saline containing 0.1% Tween-20 and 5% non-fat milk at 4 °C and incubated with the primary anti-CXCL4 and anti-CXCL12 antibodies (MAB7952 and MAB310, respectively, R&D systems) overnight. The membranes were then incubated with species-specific horseradish-peroxidase (HRP)-conjugated secondary antibodies (#31450, ThermoFisher). Following the incubation with ECL substrate (BioRad), the presence of specific proteins was determined based on chemiluminescence detected using a ChemiDoc Imaging System (BioRad). For co-IP analysis, chemokine mixtures were incubated with magnetic microbeads pre-coated with mouse anti-CXCL4 or anti-CXCL12 antibodies (R&D Systems) at 4 °C for 2 h. After incubation, microbeads were magnetically bound and washed to remove the unbound fractions. After elution, the Western blot analysis of the immune-precipitated samples was performed using goat anti-CXCL12 monoclonal antibodies (R&D Systems).

Nguyen K.T., Volkman B., Dréau D, & Nesmelova I.V. (2022). A new obligate CXCL4–CXCL12 heterodimer for studying chemokine heterodimer activities and mechanisms. Scientific Reports, 12, 17204.

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Inhibition of CXCL12-Mediated Chemotaxis

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Plerixafor (AMD3100), a CXCR4 antagonist, was reconstituted as per the manufacturer’s instructions (HY‐10046, MedChemExpress). Immune cell fractions were then preincubated with 10 µM of AMD3100 for 30 min (Biasci et al, 2020 (link)). For CXCL12 inhibition, the BOEC monolayers in the lower chambers, and immune cells in the upper chambers were per‐incubated with an CXCL12‐neutralizing antibody (25 μg/ml, MAB310, R&D Systems) for 90 min (Zheng et al, 2020 (link)). Chemotaxis assays were then performed as per described above.

Ng C., Lee K.L., Muthiah M.D., Wu K.X., Chioh F.W., Tan K., Soon G.S., Shabbir A., Loo W.M., Low Z.S., Chen Q., Tan N.S., Ng H.H., Dan Y.Y, & Cheung C. (2022). Endothelial‐immune crosstalk contributes to vasculopathy in nonalcoholic fatty liver disease. EMBO Reports, 23(6), e54271.

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Isolation and Culture of NSCLC Cells

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H460 (large cell carcinoma), A549 (adenocarcinoma), and H1299 (large cell carcinoma) NSCLC cell lines, authenticated by short tandem repeat (STR) profiling, and murine RAW 264.7, murine 3B11, and HUVECs (all purchased from ATCC) were cultured in RPMI 1640 + 10% fetal bovine serum (FBS) or in endothelial growth media (EGM-2, Lonza). The LT73 cell line was derived from a primary NSCLC adenocarcinoma and cultured in vitro in RPMI 1640 + 10% FBS.
CCR2⁺ cells were positively isolated from BM cells flushed from SCID mouse femurs using anti- CCR2⁻allophycocyanin (APC)⁺ anti-APC MicroBeads and an autoMACS Pro separator (Miltenyi Biotec). 1 × 10⁵ sorted CCR2⁺ IMs or CCR2⁻ myeloid cells were co-cultured at a 1:1 ratio with tumor cells or used to recover CM after 48 h.
Short-term NSCLC primary cultures were obtained from four early stage (I–IIb) NSCLC undergoing surgical resection using differential filtration of dissociated primary tumor tissues as described in Bertolini et al.³¹ (link)
For in vitro experiments, cells were treated with the following: peptide R (10 μM), recombinant human SDF-1α (25 ng/mL) (300-28A), cytoMCP-1 (CCL2) (25 ng/mL) (300-04), IL-8 (50 ng/mL) (200-08), GRO-α/MGSA (25 ng/mL) (300-11), and IL-6 (10 ng/mL) (200-06) (all from PeproTech); as well as neutralizing Ab anti-human/mouse SDF-1 (25 μg/mL) (MAB310) and mouse VEGF (150 ng/mL) (VEGF164) (both from R&D Systems).

Bertolini G., Cancila V., Milione M., Lo Russo G., Fortunato O., Zaffaroni N., Tortoreto M., Centonze G., Chiodoni C., Facchinetti F., Pollaci G., Taiè G., Giovinazzo F., Moro M., Camisaschi C., De Toma A., D’Alterio C., Pastorino U., Tripodo C., Scala S., Sozzi G, & Roz L. (2021). A novel CXCR4 antagonist counteracts paradoxical generation of cisplatin-induced pro-metastatic niches in lung cancer. Molecular Therapy, 29(10), 2963-2978.

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Intravitreal LPS and Anti-SDF-1 Antibody Injection Protocol

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At 1 day before laser irradiation, 10 μl LPS (10 ng/ml) was injected into the vitreous cavity from the temporal limbus of the eye by using a 32-gauge needle (Hamilton, Reno, NV) and the dose was based on previous studies in mice [8 (link)]. Because the total amount of ocular fluid was approximately 55 μl [25 (link)], the final concentration of LPS in the eye was approximately 2 ng/ml. In some eyes, immediately after CNV induction, 1 μg anti-SDF-1 antibody (MAB310; R&D Systems) was injected intravitreally from the temporal limbus of the same eye in a 10-μl volume, according to.a previous study by Otsuka et al. [26 (link)]. The control rats received the same volume of PBS 1 days before laser irradiation.

Feng Y.F., Guo H., Yuan F, & Shen M.Q. (2015). Lipopolysaccharide Promotes Choroidal Neovascularization by Up-Regulation of CXCR4 and CXCR7 Expression in Choroid Endothelial Cell. PLoS ONE, 10(8), e0136175.

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Annexin A2 Regulates BMSC Migration

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BMSC^Anxa2+/+ or BMSC^Anxa2−/− (1 × 10⁵ cells/well) were seeded onto 24-well culture plates. PCa cells or HBMECs were labeled with 2.5μg/ml of CFDA as described above. PCa cells or HBMECs were resuspended in serum-free RPMI 1640 and equilibrated for 10 min at 37°C. CFDA-labelled PCa cells or HBMECs were loaded into the top chambers of 8μm Transwell® microporous membrane 24-well plates (cat. 3422, Costar Corp, Cambridge, MA). 650μl of conditioned medium (CM) from 3 day cultures of BMSC^Anxa2+/+ or BMSC^Anxa2−/− (1 × 10⁵ cells/well) was added into the bottom well. The plates were incubated at 37°C for 3 hours. At the termination of the experiments, the intensity of fluorescence in the lower chamber, indicating the number of cells which had migrated cells, was determined by plate reader (Molecular Probes, Eugene, OR). In some cases, migration of PCa cells to CM isolated from BMSCs was analyzed in the presence of neutralizing anti-CXCL12 monoclonal antibodies (120μg/ml, cat. MAB310, R&D Systems, Minneapolis, MN) or AMD3100, a selective CXCR4 antagonist (4ng/ml, cat. A5602, Sigma, St. Louis, MO).

Jung Y., Wang J., Lee E., McGee S., Berry J.E., Yumoto K., Dai J., Keller E.T., Shiozawa Y, & Taichman R.S. (2014). Annexin 2-CXCL12 Interactions Regulate Metastatic Cell Targeting and Growth in the Bone Marrow. Molecular cancer research : MCR, 13(1), 197-207.

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Chemotactic Potency of Recombinant SDF-1α

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Chemotactic activity of the purified recombinant His-tagged protein was assessed using the Neuroprobe 96-Trans-Well Migration System (Neuroprobe, Gaithersburg, MD) and compared to commercial, non-tagged SDF-1α (R&D Systems, Minneapolis, MN) as a positive control, or a negative control lacking the presence of chemokine. 3.3×10⁶ cells/ml suspended in pre-warmed RPMI-1640 containing 20 mM HEPES and 1% BSA were placed on the upper chambers. The lower chamber contained in the same media SDF-1α at 100 ng/mL (recombinant or commercial) or no SDF-1α. The amount of cells transmigrated to the lower chamber over 3 hours at 37°C was quantified for the different conditions. To further confirm that cell migration was attributed solely to the recombinant SDF-1α, the recombinant SDF-1α was also incubated with a 14:1 molar excess of anti-SDF antibody (R&D Systems, MAB310) for 30 min at RT prior to introduction in a separate trans-well migration experiment.

Vernekar V.N., Wallace C.S., Wu M., Chao J.T., O’Connor S.K., Raleigh A., Liu X., Haugh J.M, & Reichert W.M. (2014). Bi-Ligand Surfaces with Oriented and Patterned Protein for Real-Time Tracking of Cell Migration. Colloids and surfaces. B, Biointerfaces, 123, 225-235.

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Immunofluorescence Staining of Cell Markers

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Primary antibodies used were: NCAM-1 (1:100, rabbit, Millipore, ab5032; rat, Millipore, mab310); ALCAM (1:50, goat, R&D Systems, AF1172); CNTN2 (1:50, goat, R&D Systems, AF4439); Cx40 (1:500, rabbit, Alpha Diagnostic, Cx40A); PSA (1:500, rabbit, Absolute Antibody, Ab00240-23.0); troponin T (1:100, mouse, Thermo Fisher Scientific MS-295-P0); plakoglobin (1:100, rabbit, Cell Signaling Technology, 2309S). Secondary antibodies used were: donkey anti-rabbit 555 (1:500, Thermo Fisher Scientific, A31572); donkey anti-goat 555 (1:500, Thermo Fisher Scientific, A21432); goat anti-rat 555 (1:500, Thermo Fisher Scientific, A21434); goat anti-rabbit 488 (1:500, Thermo Fisher Scientific, A11034); donkey anti-rabbit 488 (1:500, Thermo Fisher Scientific, A21206); donkey anti-goat 488 (1:500, Thermo Fisher Scientific, A11055); donkey anti-mouse 488 (1:500, Thermo Fisher Scientific, A21202); donkey anti-rat 488 (1:500, Thermo Fisher Scientific, A21208); wheat germ agglutinin (WGA) Alexa Fluor 488 conjugate (1:250, Thermo Fisher Scientific, W11261).

Delgado C., Bu L., Zhang J., Liu F.Y., Sall J., Liang F.X., Furley A.J, & Fishman G.I. (2021). Neural cell adhesion molecule is required for ventricular conduction system development. Development (Cambridge, England), 148(11), dev199431.

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Tumor Cell and CCR2+ Cell Co-Culture

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Cell cultures and reagents H460, A549, H1299 tumor cell lines, authenticated by STR profiling, murine RAW 264.7, murine 3B11 and human HUVECs endothelial cells (all purchased from ATCC) were cultured in RPMI 1640+ 10% Fetal Bovine Serum (FBS) or in endothelial grown media (EGM-2-Lonza). CCR2+ cells were positively isolated from BM cells flushed from SCID mouse femurs using anti-CCR2-APC+ Anti-APC MicroBeads and autoMACS® Pro Separator (Miltenyi). 1x10 5 sorted CCR2+ IM or CCR2-myeloid cells were co-cultured at ratio 1:1 with tumor cells or used to recover conditioned medium after 48h. Short-term NSCLC primary cultures were obtained by differential filtration of dissociated primary tumor tissue, as described in Bertolini et al [26] , from a consecutive series of Caucasian male and female of early stage (I-IIb) NSCLC patients undergoing surgical resection.
For in vitro experiments, cells were treated with: peptide R 10μM; recombinant human SDF-1α 25 ng/ml (300-28A), CytoMCP-1 (CCL2) 25ng/ml (300-04), IL-8 50ng/ml (200-08), GRO-α/MGSA 25ng/ml (300-11), IL-6 10ng/ml (200-06) (all from Peprotech) ; neutralizing Ab anti-human/mouse SDF-1 25μg/ml (MAB310) and mouse VEGF 150ng/ml (VEGF164) (all from R&D System).

Bertolini G., Cancila V., Milione M., Russo G.L., Fortunato O., Zaffaroni N., Tortoreto M., Centonze G., Chiodoni C., Facchinetti F., Giovinazzo F., Moro M., Camisaschi C., Toma A.D., D’Alterio C., Pastorino U., Tripodo C., Scala S., Sozzi G., & Roz L. (2020). SDF-1/CXCR4 inhibition prevents paradoxical generation of cisplatin-induced pro-metastatic niches.

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