Normal human CD14+ monocytes (Lonza) were cultured in α-MEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin-streptomycin, and 20 ng/ml M-CSF for 6 d to expand the precursor population and differentiate into macrophages. Cells were then detached with Accutase (A1110501; Gibco), seeded on tissue-culture plastic dishes, and cultured with M-CSF (20 ng/ml) and RANKL (20 ng/ml) for 9 d to induce multinuclear osteoclast formation, or reseeded on bone slices for bone resorption assay (Raynaud-Messina et al., 2018 (link); Zhu et al., 2020 (link)). Human osteoclasts were cultured with isotype control IgG, MMP9 function-blocking monoclonal antibody (sc-12759L; Santa Cruz), and a function-blocking antibody anti-MMP14 (DX-2400) at 100 μg/ml (Ager et al., 2015 (link); Marshall et al., 2015 (link); Zhu et al., 2020 (link)), in the presence or absence of an anti–GALECTIN-3 blocking antibody (sc-32790L; Santa Cruz) or an anti-LRP1 blocking antibody (MA1-27198; Thermo Fisher Scientific) at 25 μg/ml (Chen et al., 2015 (link); Moxon et al., 2015 (link); Seguin et al., 2017 (link)). DX-2400 was provided by the Kadmon Corporation.
Ma1 27198
Manufactured by Thermo Fisher Scientific
Sourced in Australia
The MA1-27198 is a laboratory instrument manufactured by Thermo Fisher Scientific. It is designed to perform a specific function in the laboratory setting. However, without access to the detailed technical specifications, I cannot provide a comprehensive and unbiased description of the core function of this product while maintaining the requested level of conciseness and objectivity. Therefore, the detailed description is not available.
Automatically generated - may contain errors
Lab products found in correlation
Sc 32790l, by Santa Cruz Biotechnology (3 mentions)
Normal human cd14 monocytes, by Lonza (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Ab200839, by Abcam (1 mentions)
Ratlaps ctx 1 eia kit, by Immunodiagnostic Systems (1 mentions)
Bgjb medium, by Thermo Fisher Scientific (1 mentions)
Sc 30833, by Santa Cruz Biotechnology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
D4418, by Merck Group (1 mentions)
Dt 1bon1000 96, by Immunodiagnostic Systems (1 mentions)
Wga lectin, by Merck Group (1 mentions)
Crosslaps for culture ctx 1 elisa kit, by Immunodiagnostic Systems (1 mentions)
Rankl, by R&D Systems (1 mentions)
4 protocols using ma1 27198
1
Osteoclast Differentiation and Function Assay
2
Calvarial Bone Resorption Assay
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Calvariae from 5-d-old wild-type or DKO mice were isolated aseptically, cleaned, and cultured for 16 h at 37°C in 0.5 ml of BGJb medium (Life Technologies) containing 1 mg/ml BSA (fraction V; Sigma-Aldrich; Moxon et al., 2015 (link)). Half calvariae were transferred to fresh medium with 0.1 μM parathyroid hormone (H-4835.0005; Bachem) in the presence or absence of an anti–galectin-3 monoclonal antibody (sc-32790L; Santa Cruz) or an anti-Lrp1 monoclonal antibody (MA1-27198; Thermo Fisher Scientific), and cultured for an additional 5 d. Culture supernatant was collected for bone resorption marker CTX-I level detection using RatLaps CTX-I EIA kit (AC-06F1; Immunodiagnostic Systems). The half calvariae were either fixed for immunostaining with an anti-TRAP polyclonal antibody (sc-30833; Santa Cruz), an anti–galectin-3 monoclonal antibody (#125401; Biolegend; clone M3/38), or an anti-V-type proton pump-3 (Vpp3) polyclonal antibody (ab200839; Abcam) as describe above or snap-frozen for TRAP activity assay.
3
Quantifying Bone Resorption Using Osteoclasts
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For bone resorption assays, BMDMs or pre-osteoclasts were seeded and cultured on bovine cortical bone slices (DT-1BON1000-96; Immunodiagnostic Systems) with 20 ng/ml M-CSF and 30 ng/ml RANKL (R&D Systems; Wu et al., 2017 (link); Zhang et al., 2018 (link); Zhu et al., 2020 (link)), in the presence or absence of galectin-3 (8259-GA; R&D Systems), galectin-3C (10110-GA; R&D Systems), GCS-100 (La Jolla Pharmaceutical), RAP (4480-LR; R&D Systems), an anti–galectin-3 blocking antibody (sc-32790L; Santa Cruz), or an anti-Lrp1 blocking antibody (MA1-27198; Thermo Fisher Scientific; Chen et al., 2015 (link); Demotte et al., 2010 (link); John et al., 2003 (link); Moxon et al., 2015 (link); Seguin et al., 2017 (link)). After the indicated culture period, bone samples were sonicated in PBS, stained with 20 μg/ml WGA-lectin (L3892; Sigma-Aldrich) for 45 min and then incubated with DAB tablets (D4418; Sigma-Aldrich) for 15 min. Image J software was used to quantify the resorbed area. The concentration of the CTX-I was measured using the CrossLaps for Culture CTX-I ELISA kit (AC-07F1; Immunodiagnostic Systems) according to the manufacturer’s instructions.
4
Plasma LRP1 ELISA and VSMC Experiments
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Plasma LRP1 concentrations were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) (#E91010Hu, USCN Life Sciences, Wuhan, China). This ELISA employs antibodies against a soluble extracellular LRP1 immunogen suggesting suitability for plasma analysis. Reported inter-and intra-assay variabilities are < 12%. Owing to limited plasma volumes, single measurements were taken for each patient as previously described. 19, 22 In vitro experiments Human aortic VSMC (CC-2571; Lonza, Walkersville, MD, USA) were maintained in growth medium in a humidified 5% CO 2 atmosphere at 37 C and passaged when 70e80% confluent. After five passages, LRP1-blocking antibodies or isotype control antibodies (#MA1-27198 and MA5-14453, respectively; Thermo Fisher Scientific, Scoresby, Australia) were added to the culture media at concentrations of 30 mg/mL. Cell culture supernatants were decanted after 24 hours and replaced with media containing 20 ng/mL recombinant human MMP9 (#911-MP-010; R&D Systems, Minneapolis, MN, USA). Cell culture supernatants were harvested at 24 and 48 hours, and stored at À80 C. Data presented are from three independent culture experiments.
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