The viable and dead cells were detected using a calcein-AM/PI kit (YEASEN, Shanghai, China). Briefly, RKO cells were collected and centrifuged; the supernatant was then discarded. After rinsing with assay buffer three times, the 100 µL of staining reagent was added to the cell mixture and incubated for 15 min at 37 °C.
Calcein am pi kit
Manufactured by Yeasen
Sourced in China
The Calcein-AM/PI kit is a fluorescent dye-based assay used to assess cell viability and cytotoxicity. The kit contains two dyes: Calcein-AM and Propidium Iodide (PI). Calcein-AM is a cell-permeant dye that is converted to a green-fluorescent Calcein in live cells, while PI is a red-fluorescent dye that can only enter cells with compromised membranes, indicating cell death. This kit provides a simple, reliable, and quantitative method to differentiate between live and dead cells.
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3 protocols using calcein am pi kit
1
Viable and Dead Cell Detection
2
Live/Dead Cell Staining Protocol
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Dead/live cell staining was performed using a calcein-AM/PI kit (40747ES76, Yeasen). When the confluence of r-MCs reached 90%, they were rinsed with PBS, and staining reagent (100 μL) was added for incubation for 20–30 min at 37 °C. Then, the cells were viewed under a fluorescence microscope (Axio Observe 3, Carl Zeiss, Germany). The percentage of cell death was calculated by: % of cell death = [propidium iodide-positive cells]/[calcein-AM-positive cells +propidium iodide-positive cells] × 100%.
3
3D Tumor Spheroid Viability Assay
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Live and dead cell staining was performed under a fluorescent microscope with a Calcein-AM/PI Kit (Yeasen, China). 4T1 cells at 2 × 10 3 cells per well were seeded and incubated in a 96 U-bottom plate (Thermo Fisher Scientific, USA) for 48 h to form 3D tumor spheroids. 3T3 cells were incubated with the nanoparticles for 12 h and the conditional 3T3 culture medium was added to tumor spheroids and incubated for another 12 h. The cells were stained according to the instruction of the kit. They were incubated with the kit for 25 min and washed twice with PBS. The live and dead cells were observed by using a laser confocal fluorescent microscope. The normal 3T3 culture medium mixed with the nanoparticles at the same concentration was added to 3D tumor spheroids as controls.
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