HCT116 and HT-29 cells (2.5×105) were seeded onto 6-well plates and treated for 48 h at 37°C with UA (15 µM) and/or DOX (1.5 µM). Propidium iodide (cat. no. P4170; Sigma-Aldrich; Merck KGaA) and Annexin-V-FITC Assay kit (BD Biosciences) were used to stain the cells for 30 min at room temperature in the dark according to the manufacturer's protocols. BD Accuri C6 Flow Cytometer (BD Biosciences) was used to assess the cell apoptosis. Acquisition and analysis of the data were performed using BD Accuri CFlow software (version, 1.023.1; BD Biosciences). Cells that were considered viable were FITC Annexin V and PI negative; cells that were in early apoptosis were FITC Annexin V positive and PI negative; and cells that were in late apoptosis or already dead were both FITC Annexin V and PI positive.
Annexin 5 fitc assay kit
Manufactured by BD
Sourced in United States
The Annexin V-FITC assay kit is a laboratory product used to detect and quantify apoptotic cells. It contains Annexin V, a protein that binds to phosphatidylserine, a molecule expressed on the surface of cells undergoing apoptosis. The Annexin V is conjugated with the fluorescent dye FITC, allowing for the visualization and analysis of apoptotic cells using flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Penicillin streptomycin antibiotic solution, by Thermo Fisher Scientific (2 mentions)
Accuri cflow software, by BD (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Crystal violet, by HiMedia (1 mentions)
Acridine orange, by Thermo Fisher Scientific (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Rhodamine 123, by Merck Group (1 mentions)
Caspase 3, by Merck Group (1 mentions)
X ray film, by Cytiva (1 mentions)
Bcl 2, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Coumarin 6, by Merck Group (1 mentions)
Tin 2 2 ethylhexanoate, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Sod activity, by Enzo Life Sciences (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
10 protocols using annexin 5 fitc assay kit
1
Apoptosis Assay of HCT116 and HT-29 Cells
2
Nanocarrier-Mediated Doxorubicin Delivery
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Doxorubicin (DOX), GO-201 trifluoroacetate salt (MUC1 inhibitor), Rhodamine 123 (Rh 123), Rhodamine B (RhoB), 4,6-diamidino-2-phenylindole (DAPI), Fluorescein isothiocyanate (FITC), Coumarin-6, poly ethylene glycol-5000 (PEG-5000), caprolactone, Tin (II) 2-ethylhexanoate, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), Dimethylsulfoxide (DMSO), Bradford reagent, chemiluminescence reagent purchased from Sigma aldrich (Merck, USA). Amicon ultracentrifugal filters (10 kDa), Protein estimation kit by BCA (Bicinchoninic acid) method (GeNei) were obtained from Merck Millipore (Billerica, MA, USA.) Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Trypsin, Dulbecco Phosphate Buffered Saline (PBS) and penicillin–streptomycin antibiotic solution were purchased from Gibco (Life Technologies), USA. Acridine Orange (Thermo scientific), 4% Paraformaldehyde, Crystal Violet, Radio-immunoprecipitation assay (RIPA) lysis buffer, Tris-buffered saline (TBST) were obtained from Hi Media (India). Annexin V-FITC assay kit was obtained from BD Biosciences (San Jose, USA). Primary antibodies i.e. caspase-3 and Bcl-2 were procured from Sigma Aldrich (USA), β-actin and secondary antibodies were purchased from (Santa Cruz Biotechnology, USA). X-ray films were purchased from Amersham Biosciences, UK.
3
Synthesis and Characterization of 2-Iodo-4'-methoxychalcone
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2-iodo-4′-methoxychalcone (CHA79) was synthesized as previously described [15 (link)]. Chemical reagents, such as MG, hoechst 33342, 2′,7′-dichloro-dihydrofluorescein diacetate (H2DCF-DA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), etc., were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cell culture used materials were from Thermo Fisher Scientific (Waltham, MA, USA). Annexin V-FITC assay kit was from BD Bioscience (San Jose, CA, USA). SOD activity and GSH quantitation kit were from Enzo Life Sciences (Farmingdale, NY, USA). SDS-PAGE used materials were from Bio-Rad (Hercules, CA, USA). All primary and secondary antibodies used in Western blots (WB) are listed in supplemental material (Table S1) .
4
Paclitaxel and NuBCP-9 Combination Anticancer Protocol
5
Apoptosis Induction in A549 Cells
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The apoptosis of A549 cells induced by bFGF siRNA and/or cisplatin was assessed by flow cytometry using the Annexin V-FITC Assay Kit (BD Biosciences, San Diego, CA, USA) according to the provided protocol. After incubation with Annexin V-FITC and propidium iodide, fluorescence was quantified with a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA), and the data were analyzed by CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA).
6
FITC Annexin V Apoptosis Assay
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Cells were prepared using a FITC Annexin V assay kit (BD, USA) as recommended by the manufacturer. The cells were washed twice with cold PBS and then resuspended in a 1× binding buffer to a concentration of 1 × 106 cells/mL; 100 μL of the cell suspension (containing 1 × 105 cells) was transferred to a 5 mL culture tube, and 5 μL of FITC Annexin V and 5 μL of PI were added to the tube. After gentle rotation of the cells in the tube, the cells were incubated for 15 min at room temperature (25°C) in a dark environment; then, 400 μL of the 1× binding buffer was added to each tube, and the cells were loaded onto a flow cytometer within 1 h for detection. The source data were analyzed using the FlowJo10.0.7 software.
7
Apoptosis and Inflammation Assays
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Acetylcholinesterase and phorbol 12-myristate 13-acetate were purchased from Sigma (St. Louis, MO, United States); FITC Annexin V assay kit, active Caspase-3 apoptosis kit and JC-1 were purchased from BD Biosciences (San Jose, CA, United States); IL-1β, IL-6, and TNF-α were purchased from MyBioSource (San Diego, CA, United States); IL-8 and P-selectin ELISA kits were purchased from ThermoFisher (Waltham, MA, United States); IMDM, RPMI1640, FBS, and BSA were purchased from Gibco (Waltham, MA, United States); tanshinone IIA was purchased from Shanghai Pharmaceuticals (Shanghai, China); TPO was purchased from PeproTech (Rocky Hill, NJ, United States).
8
Assessing B Cell Survival and Apoptosis
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B cell survival and apoptosis were assessed using the FITC Annexin V assay kit (BD biosciences, Heidelberg, Germany) according to manufacturers’ instructions.
9
Clausenidin Induces Apoptosis in HT-29 Cells
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HT-29 cells were seeded at a concentration of 2 × 105cells/T-25 flask in RPMI media and incubated overnight. The cells were then treated with increasing concentrations of clausenidin while the negative control was treated with 0.1 % (v/v) DMSO. Cells were harvested after treatment and washed with PBS. Annexin V assay was then carried out using FITC annexin V assay kit (BD Pharmingen, USA) following the manufacturer’s protocol and the result was analyzed on a flow cytometer.
10
IGFBPL1 Regulation of Cell Cycle and Apoptosis
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IGFBPL1 unexpressed and re-expressed KYSE150 and KYSE410 cells were starved for 12 h and then stimulated with 10% FBS for 24 h. Cells were fixed with 70% ethanol and treated using the Cell Cycle Detection Kit (KeyGEN Biotech). The cells were then analyzed by a FACS Caliber flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The same method was used to analyze KYSE510 cells before and after IGFBPL1 knockdown. Cell phase distribution was analyzed using MODFIT software (Verity Software House, ME, USA). KYSE150 and KYSE410 cells were transiently transfected with pcDNA3.1+ and pcDNA3.1+ IGFBPL1 vectors. Cell apoptosis was analyzed by FITC Annexin V assay kit according to the manufacturer’s instructions (BD Bioscience, Franklin). Apoptosis of KYSE510 cells with or without knockdown of IGFBPL1 was also analyzed. Each experiment was repeated three times.
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