Transfected cells were treated with 100 µM TMZ for 24 h, washed with Hank's D solution, harvested and counted. The eBioscience™ Annexin V Apoptosis Detection Kit APC (cat. no. 88-8007-72; Invitrogen; Thermo Fisher Scientific, Inc.) was used to measure apoptosis according to the manufacturer's protocol. A total of 1×105 cells were resuspended in 100 µl binding buffer, and 10 µl of Annexin V and 5 µl of propidium iodide (PI) were added. The cells were then incubated in the dark for 15 min at room temperature, and subsequently analyzed using an Epics Altra II cytometer (Beckman Coulter Inc., Brea, CA, USA). The data were analyzed by Kaluza analysis software (version 1.3; Beckman Coulter Inc.) and the apoptotic rate (%) was determined by adding the cell population positive for PI and annexin V (late apoptosis) and the population positive for annexin V only (early apoptosis). The experiment was repeated three times.
Epics altra 2 cytometer
Manufactured by Beckman Coulter
Sourced in United States
The Epics Altra II cytometer is a high-performance flow cytometry instrument designed for research applications. It features advanced optical and electronic systems to facilitate the detection and analysis of cells and particles. The core function of the Epics Altra II is to enable the precise measurement and characterization of various cellular properties, including size, granularity, and the expression of specific markers.
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4 protocols using epics altra 2 cytometer
1
Measurement of TMZ-induced Apoptosis
2
Quantifying Apoptosis by Flow Cytometry
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Cell apoptosis was detected using Annexin V/PI staining and flow cytometry. Cells were seeded at a density of 1.5×106 cells per well in 6-well plates. After treatment with different reagents for 48 h, the cells were collected, washed with PBS, and stained with fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide (PI) by the FITC Annexin V Apoptosis Detection Kit (BD Biosciences, Shanghai, China) according to the manufacturer's recommendations. Flow cytometric analysis was performed immediately using a Beckman Coulter Epics Altra II cytometer (Beckman Coulter, CA, USA).
3
Flow Cytometry Analysis of TfR1-Positive Cells
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Flow cytometry was used to identify TfR1-positive cells. A total of 1 × 106 cells were collected and washed in ice-cold 1 × PBS. For 30 min on ice, the cells were resuspended in 5% BSA. The cells were treated for 1 h on ice with 0.2 g per 106 cells with ABfloTM 488 rabbit anti-human CD71 mAb (A22301, ABclonal, Wuhan, China). The cells were centrifuged three times in 5 min with 1 × PBS. A total of 10,000 cells per sample were examined for fluorescence intensity on the FL1 channel using an Epics Altra II cytometer (Beckman Coulter, Miami, FL, USA) with gating to record only living cells (gate constructed from the non-antibody treatment group). FlowJo software (Tree star, Inc., Ashland, OR, USA) was used for the analysis.
4
Annexin V-FITC/PI Apoptosis Assay
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After the floating and detached cells were collected and combined, cells were treated by an Annexin V-FITC/propidium iodide apoptosis detection kit (ZP327-1, Beijing Zoman Biotechnology, Beijing, China) according to the manufacturer’s protocol. A total of 10,000 cells per sample were analyzed via Epics Altra II cytometer (Beckman Coulter, Miami, FL, USA). A Beckman Coulter Epics Altra with Expo32 software (Beckman Coulter) was used to measure cell apoptosis.
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