Upon treatments of Dictamni Cortex extracts [DC-I to DC-IV], cell proliferation of PBMC was quantified by BrdU Cell Proliferation Assay Kit (BioVision, Milpitas, CA, USA) according to the manufacturer’s protocol with optimization. Briefly, each herbal extract in various concentrations (0, 1, 10, 100, 200, 500, and 1000 μg/mL) was cultured with PBMC (1 × 105/mL) for 22.5 h. The cell culture was incubated with BrdU solution at 37 °C for 1.5 h. The cells were then incubated with fixing/denaturing solution at room temperature for 30 min, followed by incubation with BrdU Detection antibody for 1 h. The cells were washed, and anti-mouse HRP-conjugated antibody was incorporated. Upon washing, TMB substrate was added. Color development was determined at 450 nm with the VICTOR3 Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA).
Brdu cell proliferation assay kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The BrdU Cell Proliferation Assay Kit is a laboratory instrument used to measure cell proliferation. It detects and quantifies the incorporation of the synthetic nucleoside bromodeoxyuridine (BrdU) into the DNA of dividing cells. This kit provides the necessary reagents and protocols to perform the assay.
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Anti cd11c magnetic microbeads, by Miltenyi Biotec (2 mentions)
Easysep mouse cd8 t cell isolation kit, by STEMCELL (2 mentions)
Gm csf, by Merck Group (2 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (2 mentions)
Cell counting kit 8 cck 8, by Merck Group (2 mentions)
Microplate reader, by Bio-Rad (2 mentions)
Bz x700 microscope, by Keyence (2 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Keygen Biotech (2 mentions)
Victor3 multilabel plate reader, by PerkinElmer (1 mentions)
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Incucyte live cell imaging system, by Sartorius (1 mentions)
Incucyte s3 analysis software, by Sartorius (1 mentions)
Rt2 sybr green rox qpcr mastermix, by Qiagen (1 mentions)
Rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
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59 protocols using brdu cell proliferation assay kit
1
Dictamni Cortex Extracts Impact on PBMC Proliferation
2
Generating and Analyzing Murine Dendritic Cells and Macrophages
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To generate murine BMDC or BMDM, bone marrow was flushed from femurs and tibias of C57BL/6J (B6) mice and cultured in Petri dishes at 5 × 106 cells per dish using complete RPMI-1640, supplemented as described above and containing 10 ng/mL GM-CSF (MilliporeSigma) or M-CSF (culture medium supplemented with 20% L929 supernatant), respectively. GM-CSF or M-CSF were replaced on day 4. After 1 week, cells were harvested and BMDC were further purified using magnetic anti-CD11c microbeads (Miltenyi Biotec). Purified CD11c+ murine DC or BMDM were seeded into 6-well plates and matured with 200 ng/mL human IL-32 (R&D Systems) for 48 hours. DC were pulsed with 1 mg/mL OVA (EndoFit Ovalbumin, InvivoGen) or 1 nM SIINFEKL (MilliporeSigma) antigen for 24 hours. Subsequently, CD8+ T cells were purified from the spleens of OT-I mice using EasySep Mouse CD8+ T cell Isolation Kit (negative selection, STEMCELL Technologies) and cocultured with matured CD11c+ DC. T cell proliferation was assessed after 48 hours using the BrdU Cell Proliferation Assay Kit (BioVision). Purified IL-32γ matured CD11c+ DC and BMDM as well as untreated controls were also transcriptionally profiled after 24 hours using NanoString and analyzed via flow cytometry after 48 hours.
3
Cell Proliferation Assay using BrdU
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A BrdU Cell Proliferation Assay Kit (Biovision, Milpitas, CA, USA) was used to measure the cell proliferation rate according to the manufacturer’s instructions. Briefly, the cells were seeded in a 96-well plate and incubated overnight. After transfection, the cells were further cultured for 24 hrs. Then, 10 μM BrdU was added to the medium and incubated for 4 hrs. The absorbance at 450 nm was read and recorded to calculate the relative proliferation rate. The experiment was done three times with triplicate samples.
4
BrdU Cell Proliferation Assay in HepG2 and PLC5 Cells
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Cell proliferation was examined based on the incorporation of thymidine analog bromodeoxyuridine (BrdU) into newly synthesized DNA using the BrdU cell proliferation assay kit (BioVision, Mountain View, CA, USA). Briefly, cells (1 × 104 for HepG2 cells and 7.5 × 103 for PLC5 cells) were spread in 96-well plate and cultured overnight. After drug treatment for 48 h, cells were incubated with BrdU solution at 37 °C for 2 h, followed by 30 min incubation at room temperature (RT) in fixing/denaturing solution. Then, cells were incubated with BrdU detection antibody solution at RT for 1 h with gentle shaking. After washing twice, anti-mouse HRP-linked antibody solution was added to each well and the plate was placed at RT for 1 h. After washing 3 times, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added to each well and the absorbance at 450 nm was measured after color development.
5
Cell Proliferation Assay Protocols
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Cell proliferation was tested using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s instruction. CCK-8 solution was added to each well and incubated for 2 h at 37 °C and 5% CO2. The absorbance was measured at 450 nm using a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). BrdU Cell Proliferation Assay Kit (BioVision Inc.) was employed and performed according to the manufacturer’s instruction. BrdU solution was added into each well and incubated at 37 °C for 3 h and then detected by an enzyme-linked immunosorbent assay.
6
Measuring Dermal Fibroblast Proliferation
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Proliferation of dermal fibroblasts was measured using the BrdU Cell Proliferation Assay Kit (BioVision). After 4 hours of BrdU incubation, cells were fixed before adding Abs and substrate. The absorbance at 450 nm was measured. In a separate experiment, the Incucyte live-cell imaging system (Sartorius) was used to monitor cell proliferation. Cells were seeded and allowed to grow overnight. After adding different treatments, cells were monitored by Incucyte up to 5 days. Cell counts were analyzed by the Incucyte S3 Analysis software (Sartorius).
7
ZFAS1 Expression and Cell Proliferation Assay
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The RNeasy Kit, RT2 First Strand Kit, RT2 SYBR Green ROX qPCR Mastermix, and RT2 lncRNA qPCR Assay for Human ZFAS1 were purchased from Qiagen (Valencia, CA, USA). The BrdU Cell Proliferation Assay Kit was purchased from BioVision (Mountain View, CA, USA). The sorafenib was purchased from LC Laboratories (Woburn, MA, USA). The GSK-2606414 was purchased from APExBIO (Boston, MA, USA). The trans-ISRIB was purchased from Tocris Bioscience (Ellisville, MO, USA). Ceapin-A7 and 4μ8c were purchased from Sigma-Aldrich (St. Louis, MO, USA). Silencer Select pre-designed siRNA for human ZFAS1, negative control siRNA, and RNAiMAX transfection reagent were purchased from Thermo Fisher Scientific (Wilmington, DE, USA).
8
Hypoxia Effects on hUCMSCs Proliferation
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hUCMSCs (5 × 103) in 96-well plates were exposed to hypoxia for 24 h (normoxic cells served as control). Cell proliferation and viability were determined using a BrdU Cell Proliferation Assay Kit (BioVision, Milpitas, CA) and cell counting kit-8 (KeyGEN BioTECH, Nanjing, China) respectively, according to the manufacturer’s manual.
9
Cell Proliferation Assay Protocol
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Cell number was determined by a Cell Counting Kit-8 (CCK-8) (Sigma-Aldrich) following the manufacturer’s protocol. Relative cell number was normalized by the number of viable cells on day 0. BrdU (5-bromo-2′-deoxyuridine) incorporation was determined by a BrdU Cell Proliferation Assay Kit (BioVision, Milpitas, California) following the manufacturer’s protocol. BrdU incorporation rate was normalized by the cell number measured by a CCK-8 kit. Relative BrdU incorporation rate was normalized by that of control cells under matrix-attached condition.
10
BrdU Cell Proliferation Assay in HUVECs
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Cell proliferation was assessed using a BrdU Cell Proliferation Assay kit (BioVision, Milpitas, CA, USA) according to the manufacturer's instructions. In brief, HUVECs were plated in 96-well plates at a density of 6 × 104 /well in EBM-2 supplemented with EGM-2 Single Quots, and serum-starved in EBM-2 for 2 h. After serum-starvation, the cells were treated with the co-culture CM for 6 h. BrdU was then added and after 3 h incubation, BrdU incorporation into DNA was measured using a microplate reader (Bio-Rad, CA, USA).
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