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Lm609

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LM609 is a lab equipment product manufactured by Merck Group. It is designed for laboratory use, but a detailed description of its core function cannot be provided in a concise, unbiased, and factual manner without extrapolation or interpretation. The information available is limited.

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Lab products found in correlation

Alexa fluor labeled secondary antibody, by Thermo Fisher Scientific (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Facsdiva 8, by BD (2 mentions) Alexa fluor 568 labeled phalloidin, by Thermo Fisher Scientific (1 mentions) Ab32115, by Abcam (1 mentions) Nbp1 78446, by Novus Biologicals (1 mentions) Vitronectin, by BD (1 mentions) Crystal violet solution, by Merck Group (1 mentions) Fibronectin, by Merck Group (1 mentions) Image pro, by Media Cybernetics (1 mentions) Diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions) Biozero bz 8000, by Keyence (1 mentions) Flow cytometry, by BD (1 mentions) Bms 345541, by Merck Group (1 mentions) Ag1478, by Merck Group (1 mentions) Phase contrast microscope, by Leica camera (1 mentions) 24 well boyden chamber, by Corning (1 mentions) Digital camera, by Canon (1 mentions) Prolong gold with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions) Eclipse te2000 e fluorescence microscope, by Nikon (1 mentions)

31 protocols using lm609

1

Confocal Microscopy of C. glabrata Infection

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The confocal microscopy was performed as previously described [6 (link)]. Briefly, endothelial cells were grown to confluency on fibronectin-coated glass coverslips and then infected with 3x105C. glabrata cells. After 45 min, the cells were fixed with 3% paraformaldehyde and blocked with 5% goat serum containing 0.05% Triton X-100. The cells were incubated with Alexa Fluor 568-labeled phalloidin (Thermo Fisher Scientific, Cat. #A12380), rabbit anti-gC1qR antibody (Santa Cruz Biotechnology, Cat. #sc-48795), anti-integrin αvβ3 monoclonal antibody (Millipore-Sigma, clone LM609, Cat. # MAB1976), or anti-integrin αvβ5 monoclonal antibody (Millipore-Sigma, clone P1f6, Cat. # MAB1961). The C. glabrata cells were labeled with calcofluor white. After extensive rinsing, the cells were incubated with the appropriate Alexa Fluor labeled secondary antibody (Thermo Fisher Scientific, Cat. #A-11031 or A-11034), rinsed, and then imaged by confocal microscopy. Consecutive z-stacks were combined to create the final images.
Phan Q.T., Solis N.V., Lin J., Swidergall M., Singh S., Liu H., Sheppard D.C., Ibrahim A.S., Mitchell A.P, & Filler S.G. (2022). Serum bridging molecules drive candidal invasion of human but not mouse endothelial cells. PLoS Pathogens, 18(7), e1010681.
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2

Monoclonal Antibodies for Integrin Analysis

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Mouse monoclonal antibodies against the avb3 heterodimer (LM609) and b1 subunit (P5D2) were purchased from Millipore-Sigma. Specific mouse monoclonal antibodies for b3 (BV3) and aIIb (B9) subunits were bought at Origene (Rockville, MD) and Santa Cruz Biotechnology (Dallas, TX), respectively. The APClabeled goat anti-mouse IgG-specific secondary antibody was obtained from Jackson Immuno-Research (West Grove, PA).
Lacaria L., Lange J.R., Goldmann W.H., Rico F, & Alonso J.L. (2020). αvβ3 integrin expression increases elasticity in human melanoma cells. Biochemical and biophysical research communications, 525(4).
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3

Piezo1 Subunit Localization in Adherent Cells

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Cells of interest seeded on glass bottom petri dishes were fixed with 4% paraformaldehyde for 30 min at 37° and washed three times with 1× PBS. The fixed cells were permeabilized/blocked by incubation with TBST [1× tris-buffered saline and 0.1% Tween 20] containing 5% bovine serum albumin (BSA) overnight at 4°. Primary antibodies were diluted in TBST and 5% BSA solution according to the manufacturers’ recommendations and added to the cells overnight at 4°. The stained cells were washed with 1× PBS and stained with secondary antibody for 1 hour at room temperature.
Polyclonal rabbit and monoclonal mouse anti-Piezo1 was from Novus Biologicals (NBP1-78446 and NBP2-75617); both have been validated by Piezo1 knockdown/knockoff experiments (15 (link), 78 (link)). For all Piezo1 localization quantifications, the monoclonal NBP2-75617 antibody was used. Mouse anti-paxillin antibody was from Merck Millipore (MAB 3060) and rabbit anti-paxillin antibody was from Abcam (ab32115). Mouse anti–activated integrin β1 antibody was from BD Biosciences (9EG7). Rat anti–integrin αvβ3 was from Millipore (LM609).
Yao M., Tijore A., Cheng D., Li J.V., Hariharan A., Martinac B., Tran Van Nhieu G., Cox C.D, & Sheetz M. (2022). Force- and cell state–dependent recruitment of Piezo1 drives focal adhesion dynamics and calcium entry. Science Advances, 8(45), eabo1461.
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4

Integrin Blockade Impacts Virus Infection

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Cell surface α5β1 and αvβ3 integrin were inhibited in vitro by pre-treating cells with α5β1 integrin (75 μg/ml, MAB 2514) and αvβ3 integrin (10 μg/ml, LM609) blocking antibody (Millipore, Massachusetts, USA), respectively. IgG served as a control for the experiments. Following pre-treatment, cells were either infected with viruses (RSV and IAV) or treated with 25HC and MDP. For in vivo experiments, mice were treated with either IgG (control) or β1 integrin blocking antibody (anti-mouse CD29, clone-9EG7).
Pokharel S.M., Shil N.K., GC J.B., Colburn Z.T., Tsai S.Y., Segovia J.A., Chang T.H., Bandyopadhyay S., Natesan S., Jones J.C, & Bose S. (2019). Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response. Nature Communications, 10, 1482.
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5

Cell Adhesion Assay for Integrin-ECM Interactions

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To confirm the selectivity and specificity of the association between integrins and ECM ligands, a cell adhesion assay was performed using primary human retinal endothelial cells (HRECs; Cell Systems, Kirkland, WA). Briefly, 75 μl vitronectin (5 μg/ml; BD, Franklin Lakes, NJ) or fibronectin (10 μg/ml, Sigma-Aldrich; concentrations showing best adhesion to HRECs) was incubated for 1 h at 37 °C in 96-well microtiter plates. After excess ECM was removed from the wells, the uncoated area in the wells was blocked with 1% BSA. HRECs suspended at 105 cells per ml in CSC medium (Cell Systems) containing 0.1% BSA were incubated for 30 min with antibodies for α5β1 (clone JBS5, EMD Millipore, Billerica, MA) or αvβ3 (LM609, Millipore). Ten-microliter cell suspensions were inoculated into the ECM-coated wells. After incubation for 10 min at 37 °C, the cells were fixed with 10% neutral formalin (Nacalai tesque) and stained with 0.1% crystal violet solution (Sigma-Aldrich). The statistical test and variance is stated in figure legend. The stained areas were quantified with Image-Pro (Media Cybernetics, Rockville, MD).
Nakajima T., Hirata M., Shearer T.R, & Azuma M. (2014). Mechanism for laser-induced neovascularization in rat choroid: Accumulation of integrin α chain-positive cells and their ligands. Molecular Vision, 20, 864-871.
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6

Cell Migration Assay with Boyden Chambers

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To examine cell migration Boyden chamber assays were carried out using multi-well chemotaxis chambers (pore size 8 μm: BD, Franklin Lakes, NJ, USA). The mADFs or hADFs in 0.1% FBS DMEM (5 × 104 cells/well) were added to the upper chamber. 0.1% FBS DMEM with or without recombinant human CCN4 (rhCCN4; Pepro Tech, Rocky Hill, NJ) was added to the lower chamber. Six hours after plating, the upper surface of the filter of each well was rubbed with a cotton swab to remove the cells that had attached but had not migrated onto the bottom surface. The filter was fixed in 4% PFA for 10 min, stained with diamidino-2-phenylindole (DAPI: Life technologies, Waltham, USA). The number of migrated cells was counted in four randomly selected fields using fluorescence microscope (Biozero BZ-8000, Keyence Corp., Osaka, Japan).
For the inhibition assays, hADFs were pre-incubated for 30 min with either 10 ug/mL of anti-integrin α5β1 antibody (JBS5; Millipore, Temecula, CA, USA), 10 ug/mL anti-integrin αVβ3 antibody (LM609; Millipore) or with 10 μM of MEK/ERK inhibitor (PD 98059; Calbiochem, San Diego, CA, USA). Cell migration assay and western blotting assay were carried out as described.
Ono M., Masaki A., Maeda A., Kilts T.M., Hara E.S., Komori T., Pham H., Kuboki T, & Young M.F. (2018). CCN4/WISP1 controls cutaneous wound healing by modulating proliferation, migration and ECM expression in dermal fibroblasts via α5β1 and TNFα. Matrix biology : journal of the International Society for Matrix Biology, 68-69, 533-546.
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7

Integrin αvβ3 expression analysis

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A375 and MCF-7 cells were collected and suspended in phosphate- buffered saline/0.2 % bovine serum albumin. A 200 μl sample of the suspension was incubated with a mouse antihuman integrin αvβ3 mAb LM609 (Millipore, Shanghai, China) or isotype-matched control antibody DD7 (Millipore) for 45 min at 4 °C. The cells were washed three times with phosphate-buffered saline and analyzed using a flow cytometry (Becton–Dickinson, Frank- lin Lakes, NJ, USA).
Li P., Shi Y.W., Li B.X., Xu W.C., Shi Z.L., Zhou C, & Fu S. (2015). Photo-thermal effect enhances the efficiency of radiotherapy using Arg-Gly-Asp peptides-conjugated gold nanorods that target αvβ3 in melanoma cancer cells. Journal of Nanobiotechnology, 13, 52.
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8

Inhibiting EGFR and NF-κB Signaling in H. pylori Infection

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The inhibitors, AG1478 and BMS-345541, were purchased from Merck KGaA. The antibodies used are: PY99 phosphotyrosine-specific mouse monoclonal antibodies (mAb), anti-CagA rabbit polyclonal antibody, and NF-κB p65-specific mouse mAb F-6 (Santa Cruz Biotechnology); anti-EGFR rabbit mAb D38B1 (Cell Signaling); anti- α-tubulin mouse mAb B-5-1-2 (Sigma-Aldrich); goat anti-H. pylori antiserum [Kirkegaard and Perry Laboratories (KPL)]; integrin αvβ3-specific mouse mAb LM609 (Millipore); rat mAb AIIB2 (specific for integrin β1) (Hall et al., 1990 (link)) and BIIG2 (specific for integrin α5), developed by C. H. Damsky were obtained as conditioned culture media ultrafiltration concentrates from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. Before use in infection cultures, AIIB2 and BIIG2 concentrates were dialysed against PBS to remove residual antibiotics as described previously (Gorrell et al., 2013 (link)).
Tafreshi M., Guan J., Gorrell R.J., Chew N., Xin Y., Deswaerte V., Rohde M., Daly R.J., Peek RM J.r., Jenkins B.J., Davies E.M, & Kwok T. (2018). Helicobacter pylori Type IV Secretion System and Its Adhesin Subunit, CagL, Mediate Potent Inflammatory Responses in Primary Human Endothelial Cells. Frontiers in Cellular and Infection Microbiology, 8, 22.
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9

Antibody-based Immunoblotting and Immunofluorescence

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The following antibodies (Abs) were used for immunoblotting (IB): mouse mAbs to CD63 (#ab 8219), CD81 (#ab 23505), or rabbit polyclonal Abs (pAbs) to FLOTILLIN-1 (FLOT-1) (#ab 41927) from Abcam; mouse mAb to ubiquitin (sc-8017) or rabbit pAbs to ERK (#sc 93), AKT (#sc 8312), CALNEXIN (CANX) (#sc 11397), rat mAb to CD9 (#sc 18869) from Santa Cruz; rabbit pAb to ACTIN (#A2066) from Sigma and rabbit pAb to SYNAPTOPHYSIN (SYN) (#180130) from Invitrogen; rabbit pAb serum against the cytoplasmic domain of human β3 has been described (12 (link)). A rabbit mAb against β3 (#ab 75872) from Abcam was used in immunofluorescence (IF). The AP3 mAb against β3 (ATCC) was used for FACS analysis. A mouse anti-human αvβ3 integrin (VN receptor) mAb LM609 (#MAB1976) from Millipore and an isotype negative control Ab were used in adhesion and migration assays.
Singh A., Fedele C., Lu H., Nevalainen M.T., Keen J.H, & Languino L.R. (2016). Exosome-mediated Transfer of αvβ3 Integrin from Tumorigenic to Non-Tumorigenic Cells Promotes a Migratory Phenotype. Molecular cancer research : MCR, 14(11), 1136-1146.
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10

Cell Migration Assay with Inhibitor

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Cell migration was performed as previously reported [49 (link),50 (link)] using a 24-well Boyden chambers (Corning, NY, USA) with inserts of polycarbonate membranes (8µm pores). MDA-MB-231 and BT-549 cells (0.5 × 105/well) were re-suspended in 100 µl of serum-free medium in the presence or absence of different concentration of ψRGDechi (50 µM, 10 µM, 1µM), scrambled-peptide (50µM), and blocking anti-αvβ3 antibody LM609 (10 μg/mL) (Millipore, Burlington, MA, USA) and were seeded in the upper chamber. After the addition of 1% FBS or 10% FBS in the lower chamber as chemo-attractants, the trans-well were put for 24 h at 37 °C in a humidified incubator in 5% CO2. The not migrated cells were removed with cotton swabs, whereas the cells that had migrated were visualized by staining the membrane with 0,1% crystal violet in 25% methanol. Ten random fields/filter were counted under a phase contrast microscope (Leica, Wetzlar, Germany) and images were captured by a digital camera (Canon, Tokyo, Japan) attached to the microscope. All experiments were performed at least three times and the results are expressed as the percentage of migrating cells, considering the untreated control sample as 100%.
Hill B.S., Sarnella A., Capasso D., Comegna D., Del Gatto A., Gramanzini M., Albanese S., Saviano M., Zaccaro L, & Zannetti A. (2019). Therapeutic Potential of a Novel αvβ3 Antagonist to Hamper the Aggressiveness of Mesenchymal Triple Negative Breast Cancer Sub-Type. Cancers, 11(2), 139.
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