Cell suspensions were prepared at a concentration of approximately 2 × 107 cells/mL in PBS and pipetted onto glass slides coated with poly-L-lysine. The slides were immersed in a staining jar containing 4% fresh polyformaldehyde (Solarbio, Beijing, China) dissolved in PBS, and 100 µL of Proteinase K (Solarbio) was applied to each sample. To analyze cell apoptosis of spinal cord tissues, the tissues were embedded into paraffin and incubated with xylene, ethanol and Proteinase K. Subsequently, 100 µL of 1×Equilibration Buffer was added to cover the entire area of the samples, and TdT incubation buffer was added to the cells. The slides were then placed in a dark staining jar containing a solution of propidium iodide (PI, Solarbio). Excess water was removed from the slides by tapping, and 100 µL of PBS was added to maintain sample moisture. Finally, the samples were analyzed immediately under a fluorescence microscope.
Propidium iodide
Manufactured by Solarbio
Sourced in China, United States
Propidium iodide is a fluorescent dye that is commonly used in cell biology for DNA staining. It binds to DNA by intercalating between the bases and is commonly used to stain dead cells, as it can only penetrate cells with compromised cell membranes.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 fitc, by Solarbio (20 mentions)
Rnase a, by Solarbio (17 mentions)
Binding buffer, by Solarbio (8 mentions)
Calcein am, by Solarbio (7 mentions)
Facscalibur, by BD (6 mentions)
Accuri c6, by BD (5 mentions)
Cellquest software, by BD (5 mentions)
Annexin 5 fluorescein isothiocyanate, by Solarbio (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Trypsin, by Thermo Fisher Scientific (4 mentions)
Flow cytometry, by Thermo Fisher Scientific (4 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Beyotime (4 mentions)
Flow cytometry, by BD (3 mentions)
Annexin 5 fluorescein isothiocyanate annexin 5 fitc apoptosis detection kit, by Solarbio (3 mentions)
Annexin 5, by Solarbio (3 mentions)
Facscan, by BD (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
One step tunel apoptosis assay kit, by Beyotime (3 mentions)
Sulforhodamine b, by Solarbio (3 mentions)
Triton x 100 solution, by Beyotime (3 mentions)
155 protocols using propidium iodide
1
Apoptosis Analysis of Spinal Cord Tissues
2
Apoptosis Quantification in A549 and Calu-3 Cells
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A549 and Calu‐3 cells (1 × 105 per well) were seeded into 24‐well plates and cultured for three days. Cells were then resuspended in binding buffer and stained in the dark with 5 μL Annexin V‐fluorescein isothiocyanate (FITC) and propidium iodide (PI) (Solarbio) for 10 minutes. Flow cytometry was used to assess the apoptotic cells through a flow cytometer (Countstar, Shanghai, China). The apoptotic rate was expressed as the percentage of cells at the upper right quadrant in the total number of cells.
3
Megakaryocytic Cell DNA Polyploidy
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The megakaryocytic cells were seeded in 1% gelatin pre-coated 60 mm dishes at the density of 100,000 cells/dish, and performed exposure experiments as described above. When the exposure was terminated, the cells were washed with PBS twice, harvested, fixed in ice-cold 70% ethanol and kept at − 20 °C for 24 h. After rinsing with PBS twice using centrifugation (600 g, 5 min), the cells were incubated with 0.1 mg/mL RNase A (Solarbio, China) at 37 °C for 30 min, and stained with 10 μg/mL propidium iodide (PI, Solarbio, China) in the dark at room temperature for 10 min. The cells were subsequently washed twice with PBS, dispersed in 500 μL of PBS, and finally analyzed using a flow cytometer (Novocyte 1040, ACEA Biosciences, USA) under λex/λem of 535 nm/615 nm for DNA polyploidy. The percentages of cell populations with DNA ploidy of 2 N, 4 N, 8 N and 16 N were evaluated using Novo Express software.
4
Neuroprotective Mechanisms of KET and ALC
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KET hydrochloride was obtained from Gutian Medical Inc. (Fujian, China). ALC was purchased from Beijing Red Star Co. (Beijing, China). CNQX and Fura-4-AM were purchased from Sigma (USA). Dulbecco’s modified eagle medium (DMEM)/F12, B27 supplement and fetal bovine serum (FBS) were purchased from Gibco (NY, USA). MTT was from Ameresco (USA). βШ-tubulin was from Sigma (USA). Cytosine-β-D-arabinoside (Ara-C), DCFH-DA and DAPI were from Beyotime (Nanjing, China). Hoechst 33258, propidium iodide (PI) and AO-EB were from Solarbio (Beijing, China). Trizol reagent and HiFi-script cDNA Kit were purchased from CWBIO (Beijing, China). UltraSYBR Mixture (low ROX) was purchased from LEWEITECH (Shijiazhuang, China). Primary antibodies against Akt, p-Akt, CREB, p-CREB, PKA, CaMK-IV, Bcl-2, cleaved caspase-3 and horseradish peroxidase (HRP)-conjugated secondary antibodies (goat-anti-rabbit and goat-anti-mouse) were purchased from Bioss (Beijing, China). Rabbit anti-caspase-3 and BDNF IgG and mouse anti-β-actin IgG were purchased from ZSGB-BIO (Beijing, China). Primary antibody against Bax was from Proteintech (USA). Enhanced chemiluminescence was obtained from Amersham Biosciences (England, UK).
5
Apoptosis Detection by Flow Cytometry
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The cells were digested with trypsin (Solarbio, China) and fixed with 70% ethanol at 4 °C overnight. Then, 10 mg/mL RnaseA (Solarbio, China) and propidium iodide (PI) (Solarbio, China) were added and stained overnight at 4 °C. Following the instructions of the Annexin V-FITC Apoptosis Detection Kit (Solarbio, China), flow cytometry (Becton Dickinson, USA) was used to detect the apoptosis rate. Annexin V-FITC (−)/PI (−) (bottom left) were normal cells. Annexin V-FITC (+)/PI (−) cells (bottom right) were early apoptotic cells. Annexin V-FITC (+)/PI (+) (upper right) were late apoptotic cells. Annexin V (−)/PI (+) (upper left) were necrotic cells.
6
Immune Cell Activation and Response Assay
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Dulbecco’s Modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute 1640 (RPMI-1640 medium; GIBCO Co., Ltd, Shanghai, People’s Republic of China); fetal calf serum (Fumeng Biotechnical Co., Ltd, Shanghai, People’s Republic of China); pancreatin (Sagon Inc., Shanghai, People’s Republic of China); TLR4 agonist lipopolysaccharide (LPS; 0111:B4), TLR3 agonist Poly (I:C) (Sigma Inc., St Louis, MO, USA); TLR9 agonist cytosine phosphate guanosine oligodinucleotide (CpG ODN) M362 (InvivoGen Co., Ltd, Shanghai, People’s Republic of China); total protein extraction kit (Bestbio Co., Ltd, Shanghai, People’s Republic of China); human p-NF-κB antibody, human NF-κB antibody, human STAT3 antibody, human p-STAT3 antibody (Cell Signaling Technology, Inc., Beverly, MA, USA); propidium iodide (PI; Solarbio, Beijing, People’s Republic of China); RNA enzyme (Sagon Inc.); Annexin V/PI apoptosis detection (Bestbio Co., Ltd).
7
Cell Cycle Analysis by Flow Cytometry
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Cells were harvested two days after treatment with compounds or DMSO, washed twice with PBS, and fixed with 70% ethanol overnight at 4°C. Then cells were centrifuged at 3,000 rpm for 5 min, washed twice with PBS, and resuspended in PBS. After incubating with 20 μg/mL RNase A (Solarbio Beijing, China), cells were stained with 20 μg/mL propidium iodide (PI) (Solarbio Beijing, China). A Beckman Coulter flow cytometer was used for detection and FlowJo-V10 software was used to analyze the data.
8
Anticancer Effects of C. citratus Leaves
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The leaves of C. citratus was purchased from Chinese herbal medicine market in Anguo (Anguo, China). Human breast cancer MDA-MB-231 cells were obtained from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). The 0.25% trypsin and fetal bovine serum (FBS) were bought from GIBCO (Carlsbad, USA). The 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), Hoechst 33258, Propidium Iodide (PI), Annexin V-FITC/PI apoptosis detection kit and Reactive Oxygen Species assay kit were purchased from Solarbio Science & Technology Co. Ltd (Beijing, China). Bradford protein assay (BCA) kit and enhanced chemiluminescence (ECL) detection kit were provided by Beyotime Biotechnology (Shanghai, China). All antibodies were obtained from Wuhan Sanying Biotechnology (Wuhan, China). All of other chemicals and reagents were analytical grade.
9
Annexin V-FITC Apoptosis Assay
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Annexin V-fluorescein isothiocyanate (Annexin V-FITC) apoptosis detection kit (Solarbio) was utilized in this assay. In short, transfected cells were harvested and eluted with phosphate buffer solution (PBS; Solarbio). Then, 1 mL Binding Buffer (Solarbio) was used to suspend cells. Cells were orderly incubated with 5 μL Annexin V-FITC (Solarbio) and 5 μL propidium iodide (PI; Solarbio) in dark. Cell apoptotic rate was determined after cells were analyzed with flow cytometry (Thermo Fisher).
10
Cell Cycle and Apoptosis Analysis
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Targeted cells were digested with 0.25% trypsin, and 1 × 10 6 cells were immobilized with 75% cold ethanol (Solarbio, Shanghai, China) for 12 h. Cells were incubated with 50 μg/mL RNA enzymes (Solarbio, Shanghai, China) at room temperature for 1 h followed by propidium iodide (PI) (Solarbio, Shanghai, China) staining for 30 min in the dark. Flow cytometry (Bio-Rad, California, USA) was used to analyze the cell cycle. Every test was performed in triplicate.
The harvested cells were counted, and 1 × 10 6 cells were mixed with Annexin V (Solarbio, Shanghai, China) labeled with fluorescein isothiocyanate (FITC) (Solarbio, Shanghai, China) and incubated at 37°C for 10 min. Following three PBS washes for 5 min, PI staining was conducted in the dark at room temperature for 15 min, and cell apoptosis was detected by Flow cytometry. The apoptosis rate is presented as the percentage of early apoptotic cells among total cells. Every test was performed in triplicate.
The harvested cells were counted, and 1 × 10 6 cells were mixed with Annexin V (Solarbio, Shanghai, China) labeled with fluorescein isothiocyanate (FITC) (Solarbio, Shanghai, China) and incubated at 37°C for 10 min. Following three PBS washes for 5 min, PI staining was conducted in the dark at room temperature for 15 min, and cell apoptosis was detected by Flow cytometry. The apoptosis rate is presented as the percentage of early apoptotic cells among total cells. Every test was performed in triplicate.
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