Apoptosis was determined by flow cytometry analysis using the Annexin V-FITC/PI Apoptosis Kit (Becton Dickinson, Franklin Lakes, NJ, United States). Cells were seeded in 6-well plates (5 × 105 cells/well), digested with trypsin (Gibco trypsin-EDTA; Thermo Fisher Scientific), washed with phosphate-buffered saline (PBS) three times, suspended in 500 μl of binding buffer, and then incubated with 5 μl of fluorescein isothiocyanate (FITC)-conjugated Annexin V and 3 μl of propidium iodide (PI) for 15 min at room temperature in the dark. After incubation, the samples were tested using a flow cytometer (Moflo XDP, United States). Early apoptotic chondrocytes were defined as annexin V-positive and PI-negative cells, and late apoptotic chondrocytes were defined as annexin V-positive and PI-positive cells. The proportion of apoptotic cells was calculated using the FASC Calibur MT flow cytometer (BD Bioscience, NJ, United States).
Gibco trypsin edta
Manufactured by Thermo Fisher Scientific
Sourced in United States
Gibco trypsin-EDTA is a solution used for the dissociation and detachment of adherent cells in cell culture applications. It contains the enzyme trypsin and the chelating agent EDTA, which work together to break down the cell-cell and cell-matrix adhesions, allowing cells to be harvested and subcultured.
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Lab products found in correlation
Facsaria 2 flow cytometer, by BD (5 mentions)
Gibco penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Annexin 5 allophycocyanin apc dapi double staining kit, by Thermo Fisher Scientific (2 mentions)
Facsaria 2, by BD (2 mentions)
Annexin 5 allophycocyanin apc 4 6 diamidino 2 phenylindole dapi double staining kit, by Thermo Fisher Scientific (2 mentions)
Annexin 5 fitc and propidium iodide double staining kit, by Thermo Fisher Scientific (2 mentions)
Phenol red, by Thermo Fisher Scientific (2 mentions)
Annexin 5 fitc pi apoptosis kit, by BD (1 mentions)
Fasc calibur mt flow cytometer, by BD (1 mentions)
L 15 medium, by Thermo Fisher Scientific (1 mentions)
Red blood cell lysis buffer, by Merck Group (1 mentions)
Gibco fetal bovine serum, by Thermo Fisher Scientific (1 mentions)
Gibco dmem, by Thermo Fisher Scientific (1 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Calcium chloride, by Merck Group (1 mentions)
Mineral oil, by Merck Group (1 mentions)
Ethylenediamine, by Thermo Fisher Scientific (1 mentions)
Span 85, by Merck Group (1 mentions)
Gelzan cm, by Merck Group (1 mentions)
26 protocols using gibco trypsin edta
1
Annexin V-FITC/PI Apoptosis Assay
2
Establishing Patient-Derived Tumor Cell Lines
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The study was approved by the Commission Cantonale d'éthique de la recherche sur l'être humain (CER‐VD 38/15 and PB_2016‐01185); informed consent was obtained from all subjects. Experiments conformed to the principles set out in the World Medical Association‐Declaration of Helsinki and the Department of Health and the Department of Health and Human Services Belmont Report. After inking of margins and macroscopic assessment by the pathologist, part of the tumor tissue was transported to the laboratory in DMEM/F12 and mechanically and enzymatically digested as previously described. Samples were rinsed and erythrocytes lysed with Red Blood Cell Lysis Buffer (R7757, Sigma) and dissociated to single cells with 0.25% Gibco® Trypsin‐EDTA (15400‐054, Thermo Fisher Scientific Inc.) for 2 min. Trypsin was inactivated with phosphate buffer saline (PBS) 2% calf serum (CS) followed by incubation with 5 μg/ml deoxyribonuclease DNAase (1284932, Roche AG) in L‐15 medium (11415, Gibco) at 37°C for 2 min. 2% CS in PBS was added and the cells were filtered through a 70 μm pore size filter (cat# 352350, BD Falcon) and counted. Patient‐derived tumor cells were transduced with bifunctional reporter fusion gene ffLuc2/eGFP lentivirus under control of the cytomegalovirus promoter by spin infection, 25°C for 2.5 h at 400 g (Sflomos et al, 2016 ).
3
Isolation and Characterization of Glial Cells from Adult Dorsal Root Ganglia
4
Synthesis and Characterization of Doxorubicin-Loaded Nanoparticles
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Gelzan™ CM, calcium chloride, mineral oil, doxorubicin hydrochloride (Dox-HCl) and Span® 85 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Gibco™ DMEM, Gib-co™ Fetal Bovine Serum (FBS), Gibco™ Penicillin-Streptomycin, Invitrogen™ Presto-Blue™ Cell Viability Reagent and Gibco™ Trypsin-EDTA (0.05%) were purchased from Thermo Fisher Scientific (Branchburg, NJ, USA). FITC Annexin V Apoptosis Detection Kit I was purchased from BD Pharmingen™ (San Diego, CA, USA). Sodium Nitrate, hydrogen peroxide and Sodium acetate were purchased from HAYASHI PURE CHEMICAL IND. (Osaka, Japan), Ltd. (HPC). Sulfuric Acid was purchased from UNION CHEMICAL WORKS Ltd. (Hsinchu, Taiwan). Potassium permanganate and ferric chloride were purchased from SHIMAKYU’S PURE CHEMICALS (Osaka, Japan). Ethylenediamine was purchased from ACROS Organics™ (Carlsbad, CA, USA). Ethylene glycol was purchased from PanReac AppliChem (Chicago, IL, USA). Graphite powder was donated from the Department of Public Health (Taichung, Taiwan), Chung Shan Medical University. All the samples in this study were sterilized in 70% (v/v) ethanol solution at 25 °C for 24 h. Next, samples were washed three times in sterile phosphate buffered saline (PBS) for five minutes each time. Finally, samples were dried or used directly for testing.
5
Annexin V-APC/DAPI Apoptosis Assay
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After detecting apoptosis by flow cytometry, we used an annexin V-allophycocyanin (APC)/DAPI double-staining kit (Thermo Fisher Scientific) to analyze cellular apoptosis. Cells were seeded in six-well plates (5 × 105 cells/well) and then digested with trypsin (GIBCO trypsin-EDTA; Thermo Fisher Scientific), washed with PBS three times, suspended in 500 μL binding buffer, and then incubated with 5 μL fluorescein isothiocyanate (FITC)-conjugated annexin V and 3 μL propidium iodide (PI) for 15 min at room temperature in the dark. The stained cells were detected using the BD FACSAria II flow cytometer (BD Biosciences, Hercules, CA, USA).
6
Apoptosis Evaluation in PC9R Cells
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An annexin-V-allophycocyanin (APC) and 4′,6-diamidino-2-phenylindole (DAPI) double staining kit (Thermo Fisher Scientific) was used to evaluate apoptosis. The transfected PC9R cells were seeded in 6-well plates (5 × 105 cells/well) and were treated with 1 μM gefitinib. Cells were then digested with trypsin (Gibco® Trypsin-EDTA, Thermo Fisher Scientific, MA, US), washed with PBS three times, suspended in 500 μl of binding buffer, and were then incubated with 5 μl APC-conjugated annexin-V and 3 μl DAPI for 15 min at room temperature in the dark. The stained cells were detected using the BD FACS Aria II flow cytometer (BD biosciences, CA, USA).
7
Apoptosis Detection by Flow Cytometry
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Detecting apoptosis by flow cytometry An annexin V-allophycocyanin (APC)/DAPI double-staining kit (Thermo Fisher Scientific) was used to analyze cellular apoptosis. Cells were seeded in 6-well plates (5 × 105 cells/well) and then digested with trypsin (Gibco trypsin-EDTA, Thermo Fisher Scientific), washed with PBS three times, suspended in 500 μL of binding buffer, and then incubated with 5 μL of fluorescein isothiocyanate (FITC)-conjugated annexin V and 3 μL of PI for 15 min at room temperature in the dark. The stained cells were detected using the BD FACS Aria II flow cytometer (BD Biosciences, Hercules, CA, USA).
8
siRNA-Mediated Knockdown of PCSK9 in HaCaT Cells
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For siRNA-mediated knockdown experiments, 3 parallel transfections were conducted using the SMARTpool siRNAs (mixture of 4 siRNAs), siRNA targeting PCSK9, and negative control siRNA, which were obtained from Dharmacon-Horizon Discovery. After overnight incubation, HaCaT cells (47 (link)) were transfected with 20 pmol of SMARTpool siRNAs using Lipofectamine RNAiMax Reagent (Thermo Fisher Scientific) as per the manufacturer’s instructions. After 48 hours, cells were washed with PBS and were harvested for RNA extraction using a mixture of Gibco Trypsin-EDTA (0.5%) and no phenol red (Thermo Fisher Scientific). Trypsin was neutralized with FBS and the cells were collected by centrifugation and resuspended in 100 μL of RNAlater. qPCR analysis was performed to test the efficiency of the transfection. Further expression analysis was performed using RNA-Seq as described above.
9
Analyzing Apoptosis in PC9R Cells
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An Annexin V-APC and DAPI double staining kit (Thermo Fisher Scientific, Inc.) was used to analyze cellular apoptosis. Transfected PC9R cells were seeded in 6-well plates (5×105 cells/well) and treated with 1 µM gefitinib. Cells were then digested with trypsin (Gibco® trypsin-EDTA; Thermo Fisher Scientific, Inc.), washed with PBS three times, suspended in 500 µl binding buffer and then incubated with 5 µl APC-conjugated Annexin V and 3 µl DAPI for 15 min at room temperature in the dark. The stained cells were detected using a BD FACSAria II flow cytometer (BD Biosciences, San Jose, CA, USA).
10
Transfection of HEK 293 Cells with Glycine Receptor Variants
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