The GBC-SD cells and SGC996 cells were transfected with the miR-141 inhibitor or miR control inhibitor and added to each well of a 96-well plate for 48 h. After that, the cells were collected and dyed with an Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) (Roche, Mannheim, Germany) at room temperature for 15 min in the dark following with the manufacturer's protocols. Then, the cell apoptosis was detected with flow cytometry.
Annexin 5 fluorescein isothiocyanate fitc
Manufactured by Roche
Sourced in Germany
Annexin V-fluorescein isothiocyanate (FITC) is a biochemical reagent used in laboratory applications. It binds to phosphatidylserine, a phospholipid that is externalized during the process of apoptosis or cell death. The FITC label allows for the detection and quantification of apoptotic cells through fluorescence-based techniques, such as flow cytometry.
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Lab products found in correlation
3 protocols using annexin 5 fluorescein isothiocyanate fitc
1
Apoptosis Assay of Transfected Cells
2
Tannins-Enriched Fractions of Emblica officinalis
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Tannins enriched fractions of E. officinalis (EOT) fruits were provided as gift samples by Indian Herbs Research and Supply Company (Saharanpur, India). The tannins were extracted as per the method given by Ghosal et al. (1996) which involved deactivating the hydrolytic enzymes present in the fresh juice of E. officinalis, following which, column chromatography was performed over Sephadex LH-20 employing methanol and methanol-water as the eluent. The concentration of the various tannins was as follows: emblicanin A (37%), emblicanin B (33%), punigluconin (12%), pedunculagin (14%), rutin (3%), and gallic acid (1%) (Ghosal et al., 1996 ). Piracetam (PCT) was provided as gift sample by Arbro Pharmaceuticals Ltd. (Delhi, India). ATP bioluminescence assay kit and JC-1 (5, 5, 6, 6- tetrachloro-1,1,3,3- tetraethyl benzimidazolyl carbocyanine iodide) were obtained from Sigma–Aldrich (India). BCA protein assay kit was procured from Span Diagnostics Limited, Gujarat, India. Annexin V– fluorescein isothiocyanate (FITC) and ApoDirect kits were purchased from Roche Inc. All other reagents used in the experiments were of analytical grade. Double-distilled water was used throughout the experimental work.
3
Annexin V-FITC/PI Apoptosis Assay
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Cancer cells (5 × 105 cells/well in a 24-well culture plate) were treated at IC0, IC20 and IC50 concentrations. After incubation at 37 °C under a 5% CO2 atmosphere for 24 h, the cells were washed twice with phosphate-buffered saline (PBS) and centrifuged. Next, the cells were resuspended and stained with 100 μL of binding buffer containing five μL of annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) (Roche Diagnostics, Mannheim, Germany) for 15 min in the dark. The fluorescence intensity of the cells was measured. The dot plot character of the cells was processed using a flow cytometer (Beckman Coulter, IN, USA). The percentage of viable and apoptotic cells was calculated [23 (link)].
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