The ALP activity assay is widely used to estimate the early osteogenesis ability of stem cells. BMSCs were plated in 6-well plates at a density of 1.0 × 105 cells per well and treated with osteogenic inducing medium containing different concentrations of puerarin (0, 10−4, 10−5, 10−6 and 10−7 mol/L). After 7 and 14 days of induction, cells were rinsed three times with PBS and solubilized in the lysis solution (ripa buffer and PMSF mixing in a ratio of 99 to 1) (Solarbio, Beijing, China) for 15 min on ice. Lysed the collected solution under ultrasound for 10 cycles (Bioruptor Pico, Diagenode, Belgium), and then, cell lysates were centrifuged at 12,000 × g for 5 min at 4 ℃. The supernatant was collected to obtain protein. Following the instructions of the manufacturer, an ALP activity assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to measure the absorbance of the samples using a microplate reader at 520 nm. ALP activity was normalized to the respective total protein concentration detected by the bicinchoninic acid (BCA) protein assay kit (Solarbio, Beijing, China).
Lysis solution
Manufactured by Solarbio
Sourced in China
Lysis solution is a reagent used in molecular biology and biochemistry to disrupt the cell membrane and lyse cells, releasing their contents for further analysis. It is a key component in various cell extraction and sample preparation protocols.
Automatically generated - may contain errors
Lab products found in correlation
Bioruptor pico, by Diagenode (1 mentions)
Bicinchoninic acid bca protein assay kit, by Solarbio (1 mentions)
Alp activity assay kit, by Nanjing Jiancheng (1 mentions)
Dynabeads mouse cd4 kit, by Thermo Fisher Scientific (1 mentions)
Pe conjugated rat anti mouse cd8a, by BD (1 mentions)
Fitc conjugated rat anti mouse cd4, by BD (1 mentions)
Cd8 t cell, by BioLegend (1 mentions)
Bca kit, by Beyotime (1 mentions)
5 protocols using lysis solution
1
Puerarin Enhances Osteogenic Potential
2
Isolation of Murine Splenic CD4+ T Cells
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Single-cell spleen suspensions were prepared as previously described.31 (link) In brief, the spleen tissues were cut into small pieces and grinded gently with the plunger of a 5-mL syringe until single-cell suspensions were obtained. Samples were filtered through nylon mesh to remove debris, then the spleen cell suspensions were centrifuged at 300 ×g for 10 min at 4°C. The erythrocytes in cell suspensions were eliminated by a lysis solution (Solarbio, Beijing, China). The CD4+ T cells from the spleen single-cell suspensions were isolated by negative selection using the Dynabeads™ Mouse CD4 Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The purity of CD4+ T cells was above 90% as detected by flow cytometry.
3
Murine Lymphocyte Phenotyping by Flow Cytometry
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One week after the final immunization, blood was collected from five mice in each group and poured slowly into anticoagulation tubes. Staining of the cells for flow cytometry analysis was performed by direct staining. Briefly, the cells were stained with optimal concentrations of APC-conjugated Hamster anti-mouse CD3e (0·2 mg mL−1, clone 145–2C11, BD Biosciences, San Jose, CA, USA), FITC-conjugated Rat anti-mouse CD4 (0·5 mg mL−1, clone GK 1·5, BD Biosciences, San Jose, CA, USA), and PE-conjugated Rat anti-mouse CD8a (0·2 mg mL−1, clone 53–6·7, BD Biosciences, San Jose, CA, USA), and incubated at room temperature for 30 min. Red blood cells were lysed by the addition of a triple volume of lysis solution (Solarbio, Beijing, China) on ice for 15 min. Cells were then washed twice in PBS, suspended in 300 µL PBS, and immediately analysed with a BD FACSCalibur™ flow cytometer (BD Biosciences, Heidelberg, Germany).
4
Tonsil Mononuclear Cell Isolation
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Different subtypes of mononuclear cells in the tonsil of each participant were separated by gradient centrifugation at 400 × g for 30 min at 4°C and enriched using magnetic beads. In brief, the tonsil tissues were cut into small pieces and ground gently with the plunger of a 5 ml syringe until single-cell suspensions were obtained. Samples were filtered through nylon mesh to remove debris, then the tonsil cell suspensions were centrifuged at 300 × g for 10 min at 4°C. The erythrocytes in the cell suspensions were eliminated using a lysis solution (Solarbio, Beijing, China). The CD4+ T cells, CD8+ T cells, B cells and monocytes from the tonsil single-cell suspensions were isolated by using the MojoSort™ Human CD4 T cell, CD8 T cell, pan B cell, and Pan Monocyte Cell Isolation Kit (Cat No. 480009, 480011, 480081, 480059, BioLegend Inc. San Diego, CA, USA), respectively, according to the manufacturer’s instructions.
5
Endometrial Protein Expression Analysis
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One piece of endometrial tissue from each of the three groups was removed and rinsed repeatedly with PBS. Lysis solution (Solarbio, China) was prepared according to the instructions to lyse the cells. After centrifugation for 30 min (conditions: 12,000 r/s, 4 °C), 3 µl of protein supernatant was collected to determine the protein concentration. Protein concentration was determined according to a BCA kit (Beyotime Institute of Biotechnology, China), and protein denaturation was performed after sample loading. After gel pouring, pre-electrophoresis, and sample loading, the pressure line was set to a constant voltage of 30 V for 60 min, and the sample was visualized as a narrow line between the two separate layers of gel. Then, the electrophoresis apparatus was set to a constant voltage of 100 V for electrophoresis. After film transfer, the membrane was placed in 5 % milk for blocking for ≥ 1 h. The membrane was incubated with the corresponding primary antibody and shaken at 4 overnight. The secondary antibody was added, shaken at room temperature for 1 h, and rinsed with TBST. The membrane was removed, and developer solution was added to develop the film.
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