Tendon-derived stromal cells isolated from healthy hamstring and diseased supraspinatus were seeded at a density of 30,000 cells per well in a 12-well plate (mRNA) or 60,000 cells in a 6-well plate (flow cytometry). Cells were allowed to reach 80% confluence prior to stimulation with IL-1β (10 ng/mL-1). Tendon cells were incubated in DMEM F12 medium (Lonza) containing 1% heat-inactivated human serum (Sigma). Medium containing sterile filtered 0.1% endotoxin-free BSA (Sigma) diluted in PBS was used for vehicle-only controls. After IL-1β or vehicle treatment, cells were incubated for 24 hours at 37 °C and 5% CO2 until harvest of the lysate for mRNA or flow cytometry.
Endotoxin free bsa
Manufactured by Merck Group
Endotoxin-free Bovine Serum Albumin (BSA) is a purified protein product derived from bovine serum. It is a highly versatile laboratory reagent used in a variety of applications, including cell culture, immunoassays, and protein stabilization. The endotoxin-free nature of this BSA ensures minimal interference with experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Heat inactivated human serum, by Merck Group (3 mentions)
Il 1β, by Merck Group (3 mentions)
Dmem f12 medium, by Lonza (2 mentions)
Phenol red free dmem f12 medium, by Thermo Fisher Scientific (2 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Dmem media, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Sodium phosphate dibasic, by Merck Group (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
O phenylenediamine, by Merck Group (1 mentions)
Trimethylsilyldiazomethane, by Merck Group (1 mentions)
Arachidonate, by Merck Group (1 mentions)
Cell culture reagents, by Thermo Fisher Scientific (1 mentions)
8 protocols using endotoxin free bsa
1
Tendon Cell Inflammatory Response Assay
2
BAFF-R Cell Line Maintenance Protocol
3
Measuring Eosinophil Peroxidase Activity
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Cultured eosinophils were suspended in RPMI 1640, no phenol red (Life Technologies) containing 0.5% endotoxin-free BSA (Sigma-Aldrich, St. Louis, MO) at 1×106 cells per mL, and a volume of 0.1 mL per well was transferred to a 96-well plate. Cells were incubated at 37°C for 30 minutes, and then phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) was added at a concentration of 100 or 500 ng per mL and incubated for an additional 2 hours. OMB substrate buffer (0.05 M) was freshly prepared for each assay and contained 0.2 mg per mL o-phenylenediamine, 0.0243 M citric acid, 0.0514 M dibasic sodium phosphate, and 0.00012% H2O2 (Sigma-Aldrich). OMB substrate buffer (100 µL) was added to each well, and supernatants were incubated at room temperature for 10–15 minutes. As a positive control for the assay, total EPO activity of unstimulated cells (no PMA) was assayed as above with the addition of 0.01% Triton X-100 (Thermo Scientific). The colorimetric reaction was stopped with the addition of 50 µL per well of 2 N H2SO4, and absorbance (492 nm) was measured on a Synergy 2 plate reader. Data are presented as the fold change (mean ± SEM, n = 4 independent experiments with 3 wells per condition per experiment) in EPO activity detected in the supernatants of stimulated cells compared to unstimulated cells on the same plate.
4
Mouse Model of Protein Overload
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The mouse model of protein overload was established by intraperitoneal (i.p.) injection of BSA as previously described [42 (link), 43 (link)]. KO mice and control littermates received endotoxin-free BSA (Sigma, St. Louis, MO) (250mg/ml, dissolved in PBS) or an equal volume of PBS intraperitoneally for 7 consecutive days with increasing doses (2, 4, 6, 8, 10, 10, 10 mg/g body weight). Additional wild type FVB mice were treated with endotoxin-free BSA via i.p. injection as described above and received i.p. injection of Trig (1mg/kg, Sigma), SF (12.5 mg/kg, Sigma) or vehicle every the other day. Spot urine was collected before injection and 7 days after first injection and subjected to urinary albumin assay adjusted with urine creatinine concentrations. Proteinuria was confirmed by urine electrophoresis followed by Coomassie Brilliant Blue staining. Mice were sacrificed on day 7.
5
Bioactive Lipid Mediator Profiling in Tendinopathy
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As IL-1β induces NF-κB target genes known to be highly expressed in early stage tendinopathy9 (link), we investigated bioactive pro-resolving lipid mediator profiles in cells derived from healthy and diseased human tendons in the presence of IL-1β. Tendon-derived stromal cells from healthy hamstring (n = 8) and diseased supraspinatus tendons (n = 8) were seeded at a density of 60,000 cells per well in a 6 well plate. Tendon cells were allowed to reach 80% confluence prior to stimulation with IL-1β (Merck, 10ngml—1) in DMEM F12 phenol red free medium (Gibco) containing 1% heat inactivated human serum (Sigma) and 1% penicillin-streptomycin. Non-treated cells (vehicle only, containing 0.1% endotoxin free BSA, Sigma) served as controls for each experiment. After treatment, cells were then incubated at 37 ° C and 5% CO2 until harvest of the media and lysate for bioactive lipid mediator profiling after 24 h.
6
Mass Spectrometry Lipid Analysis
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Trimethylsilyl-diazomethane (as a 2 M solution in hexanes), arachidonate, fatty acid free (FAF)- and endotoxin free- BSA were from Sigma–Aldrich. 1-heptadcanoyl-2-hexadecanoyl-sn-glycero-3-(phosphoinositol-3,4,5-trisphosphate) (C17:0/C16:0-PI(3,4,5)P3, as a hepta-sodium salt) used as an internal standard for mass spectrometry (ISD), was made at the Babraham Institute as previously described (Clark et al., 2011 (link)). All cell culture reagents were from Invitrogen, while all other reagents, unless specified, were from Sigma.
7
Comparative Analysis of Tendon Cell Responses
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Tendon-derived stromal cells from healthy hamstring (n = 5) and diseased supraspinatus tendons (n = 5) were seeded at a density of 15,000 cells per well in a 24 well plate. Tendon cells were allowed to reach 80% confluence prior to stimulation with IL-1β (Merck, 10ngml—1) in DMEM F12 phenol red free medium (Gibco) containing 1% heat inactivated human serum (Sigma) and 1% penicillin-streptomycin. Non-treated cells (vehicle only, containing 0.1% endotoxin free BSA, Sigma) served as controls for each experiment. After cytokine treatment, cells were then incubated at 37 °C and 5% CO2 until harvest of the cell lysate for mRNA after 24 h. For tissues, samples of healthy subscapularis (n = 4) and diseased supraspinatus tendons (n = 14) were snap frozen and stored at −80 °C. RNA isolation, cDNA synthesis and quantitative PCR were performed using previously published protocols9 (link). Pre-validated Qiagen primer assays (15-PGDH, STAT-1, IL6, PDPN, β-actin and GAPDH) were used for qPCR. Results were calculated using the ddCt method using reference genes for human β-actin and GAPDH. Results were consistent using these reference genes and data are shown normalized to β-actin.
8
Prostaglandin Profiling in Tendinopathy
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As IL-1β induces nuclear factor kappa beta (NF-κB) target genes known to be highly expressed in early-stage tendinopathy [18 (link)], we performed profiling of prostaglandins in cells derived from healthy and diseased human tendons in the presence and absence of IL-1β. Tendon-derived stromal cells from healthy hamstring (n = 8), diseased supraspinatus tendons (n = 8), and diseased Achilles tendons (n = 6) were seeded at a density of 60,000 cells per well in a six-well plate. Tendon cells were allowed to reach 80% confluence prior to treatment with IL-1β (Merck, 10 ng/mL) in DMEM F12 medium (Lonza, UK) containing 1% heat-inactivated human serum (Sigma) and 1% penicillin-streptomycin. Non-treated cells (vehicle only, containing 0.1% endotoxin-free BSA, Sigma) served as controls for each experiment. After treatment, cells were then incubated at 37 ° C and 5% CO2 until harvest of the supernatant for prostaglandin profiling after 24 h. Samples were stored at − 80 °C prior to analysis.
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