The cyclic stretching device (Model ST-140; STREX Co., Ltd., Osaka, Japan) was used to generate stretching loading to tenocytes in vitro. Cells (1× 105 cells) were seeded onto elastic silicone chambers (2 cm × 2 cm) that had been pre-coated with 5 μg/mL rat tail type I collagen (Hangzhou shengyou Biotechnology Co., Zhejiang, China) and grown to confluence overnight. Cells were subjected to uniaxial cyclic stretching (10%, 0.5 Hz, 1 h). For stretching experiments with inhibitors (0.5 μM Lat A, 10 μM ANA, 10 μM GSK-J4 and 10 μM Y27632; MedChemExpress, Monmouth Junction, NJ, USA), cells were preincubated for 1 h prior to stretching, and the stretching was performed in media with inhibitors added. As a control, static cells were cultured in a chamber under the same conditions without any stretching.
Lat a
Manufactured by MedChemExpress
Sourced in United States
Lat A is a lab equipment product from MedChemExpress. It is a small molecule that functions as an actin polymerization inhibitor.
Automatically generated - may contain errors
2 protocols using lat a
1
Cyclic Stretching of Tenocytes In Vitro
2
Macrophage polarization by HNSCC and BMSC sEVs
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Macrophages (1 × 106 cells/well) were seeded into six‐well plates and cocultured with 50 μg sEVs derived from HN6, HN6NT5EKO, SCC25, SCC25NT5EKO, BMSC, and BMSCNT5EOE. The experiments were divided into seven groups: M0, M2, M2+sEVsHNSCC, M2+sEVsHNSCC‐NT5EKO, M2+sEVsBMSC, M2+sEVsBMSC‐NT5EOE, and M2+sEVsBMSC‐NT5EOE + Lat A (30 μM, MedChemExpress, Monmouth Junction, NJ, USA). The CM was composed of RPMI1640 medium without FBS and was collected after incubation for 24 h. After centrifugation at 10,000×g for 10 min at 4°C, the coculture supernatant was added to a 96‐well plate to detect human IL‐6, IL‐10, TNF‐α, and TGF‐β1 using a human IL‐6 enzyme‐linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA), human IL‐10 ELISA kit (R&D Systems), human TNF‐α ELISA kit (R&D Systems), and human TGF‐β1 ELISA kit (R&D Systems) according to the manufacturer's instructions.
The procedure for extracting sEVs from serum samples has been previously described in 2.6. A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.
The procedure for extracting sEVs from serum samples has been previously described in 2.6. A human CD73 ELISA kit (R&D Systems) and ELISA ancillary reagent kit (R&D Systems) were used to measure the CD73 level in sEVs from the serum.
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