An Alexa Fluor 488 Annexin V and PI kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used to assess apoptosis. Briefly, 1×105 U373-MG cells were harvested, washed twice with cold PBS and resuspended in 100 µl binding buffer (Thermo Fisher Scientific, Inc.). A total of 5 µl Annexin V-fluorescein isothiocyanate and 1 µl PI were added to the solution. Following 15 min incubation at 4°C, 400 µl binding buffer was added to the solution and cells were analyzed using the Accuri™ C6 Flow Cytometer. The results were analyzed using CellQuest™ software 1.0 (BD Biosciences). A quadrant dot plot was used to identify whether cells were in the early or late phase of apoptosis and whether they were living or necrotic, as previously described (24 (link)).
Alexa fluor 488 annexin 5 and pi kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor 488 Annexin V and PI kit is a fluorescent labeling kit used for the detection of apoptosis in cells. It contains Alexa Fluor 488-conjugated Annexin V and propidium iodide (PI) for the identification of early and late apoptotic cells.
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2 protocols using alexa fluor 488 annexin 5 and pi kit
1
Assessing Apoptosis in U373-MG Cells
2
Flow Cytometry-Based Apoptosis Assay
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Cell apoptosis was assessed using Annexin V-FITC and PI fluorescent staining (Alexa®Fluor 488 Annexin V and PI Kit, no.V13241, Invitrogen, USA). Briefly, Mac-ASCs and Sat-ASCs were collected by centrifugation and washed once with PBS. Approximately 105 cells in 1 mL of PBS were subsequently stained with 5 μL Annexin V-FITC and 1 μL PI for 20 min at room temperature. Blank control, and Annexin V and PI single staining controls were assessed as well. Flow cytometry data were analysed with the ModFit software (version 4.0, Verity Software House Inc., Topsham, ME, USA).
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