Calcein pi assay kit

Manufactured by Beyotime

Sourced in China

The Calcein/PI assay kit is a laboratory tool used to assess cell viability and cytotoxicity. It utilizes the fluorescent dyes Calcein and Propidium Iodide (PI) to differentiate between live and dead cells. Calcein is a cell-permeant dye that stains live cells, while PI is only able to penetrate damaged cell membranes, staining the nuclei of dead cells. This kit provides a simple and reliable method to quantify cellular health in a variety of experimental settings.

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Spelling variants of this product

Calcein/PI Cell Viability/Cytotoxicity Assay Kit (65 citations) Calcein/PI Live/Dead Viability/Cytotoxicity Assay Kit (13 citations) Calcein/PI cell viability assay kit (8 citations) Calcein/PI Live/Dead Viability Assay Kit (8 citations) Calcein (6 citations) Calcein-AM/PI Cell Viability/Cytotoxicity Assay Kit (5 citations) Calcein/PI Live/Dead Assay kit (3 citations) Calcein-AM/PI Cell Live/Dead Assay Kit (3 citations) Calcein/PI Cell Viability and Cytotoxicity Assay Kit (2 citations) Calcein/PI cell activity and cytotoxicity assay kit (2 citations)

6 protocols using calcein pi assay kit

Cell Viability and Cytotoxicity Assay

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Cell viability was measured using the Cell Counting Kit-8 (CCK-8) (Solarbio, Beijing, China) according to the manufacturer's instructions. In brief, 1 × 10⁴ cells were incubated with 10 μL CCK-8 reagent for 30 min, and optical density (OD) values at 450 nm were measured.
A calcein/PI assay kit (Beyotime, Shanghai, China) was used to simultaneously stain viable and dead cells. Calcein AM/PI assay solution was prepared according to the manufacturer's protocol. The cells were washed twice with PBS and incubated with Calcein AM/PI assay solution at 37 °C for 15 min. Fluorescence was detected using an ECLIPSE Ni fluorescence microscope (Nikon). Cell counting was performed using the ImageJ software.

Tian X., Yan X., Zang N., Duan W., Wang T., Li X., Ma L., Chen L., Chen J, & Hou X. (2024). Injectable thermosensitive selenium-containing hydrogel as mesenchymal stem cell carrier to improve treatment efficiency in limb ischemia. Materials Today Bio, 25, 100967.

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Detailed Reagents and Materials Protocol

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Glucose, streptozocin (STZ), and Evans blue were purchased from Sigma-Aldrich (St. Louis, MO, USA), while advanced glycosylation end products (AGEs) were acquired from BioVision (Palo Alto, CA, USA). Tunicamycin (TUN) was obtained from Abcam (Cambridge, CA, USA), while 4-phenylbutyric acid (4-PBA), BAPTA-AM, and Protein A/G magnetic beads were purchased from MedChemExpress (Lowell, NJ, USA). Cell counting kit-8 (CCK8), JC-1 assay kit, Calcein/PI assay kit, MPTP assay kit, Mito-Tracker Green, ER-Tracker Red, and a mitochondria isolation kit were purchased from Beyotime (Nantong, China). On the other hand, the TUNEL assay kits were purchased from Roche (Basel, Switzerland). MitoSOX™ Red, DAPI, ECL kits, and goat anti-mouse/rabbit IgG (H+L, Alexa Fluor 555/488) were acquired from Thermo Fisher (Waltham, CA, USA). We used primary antibodies including GRP75, Cyt c, Bcl-xl, Bax, cleaved caspase-3, and CHOP (Cell Signaling Technology, Boston, MA, USA); 4-HNE and Brn3a (Abcam, Cambridge, CA, USA); IP3R1, VDAC1, VEGF, 8-OHDG, and 3-NT (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and COX IV, Bcl-2, and β-actin (Proteintech, Wuhan, China). More detailed information about the materials and reagents is offered in Table S1.

Li Y., Li H.Y., Shao J., Zhu L., Xie T.H., Cai J., Wang W., Cai M.X., Wang Z.L., Yao Y, & Wei T.T. (2022). GRP75 Modulates Endoplasmic Reticulum–Mitochondria Coupling and Accelerates Ca2+-Dependent Endothelial Cell Apoptosis in Diabetic Retinopathy. Biomolecules, 12(12), 1778.

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Chondrocyte Viability Assay with FLP

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The normal and injured chondrocytes were seeded in a 24-well culture plate at a density of 1 × 10⁵ cells per well and cultured overnight. 50 μg/mL and 200 μg/mL FLP were then co-cultured with these chondrocytes for 48 h at 37 °C, respectively. The cells were washed and fixed with paraformaldehyde, followed by the incubation with fluorescent live/dead staining (Calcein/PI assay kit, Beyotime). The region of interest was captured by a fluorescent microscope (Olympus), and the mean intensity of the fluorescence (mean fluro-intensity) was measured with Image J software.

Zhang X., You Y., Sun Y., Guo X., Han L.i.n., Zong M, & Shi J. (2022). Catalytic anti-oxidative stress for osteoarthritis treatment by few-layered phosphorene. Materials Today Bio, 17, 100462.

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Quantifying Live and Dead Cells

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A total of 1 × 10⁵ cells were seeded in ultra-low-attachment 96-well plates and incubated for 24 hours. The Calcein/PI Assay Kit (Beyotime, Shanghai, China) was used to analyze the levels of living and dead cells. The plate was centrifuged at 400 × g for 5 minutes, and the cells were washed once with PBS. Cells were stained with 100 μL of Calcein AM/PI (1:1) in the dark for 30 minutes at 37°C. Cell immunofluorescence was performed using the Electronic Ballast EBQ 100–04 (Leistungselektronik JENA GmbH, Jena, Germany). Living cells appeared green, while dead cells appeared red in the fluorescent images.

Cao L., Zhang S., Peng H., Lin Y., Xi Z., Lin W., Guo J., Wu G., Yu F., Zhang H, & Ye H. (2024). Identification and validation of anoikis-related lncRNAs for prognostic significance and immune microenvironment characterization in ovarian cancer. Aging (Albany NY), 16(2), 1463-1483.

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Evaluating Cell Proliferation and Viability

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To assess the effects of materials on cell proliferation and viability, Cell Counting Kit-8 (CCK8), Calcein/PI staining, and EdU fluorescence analysis were performed.
For CCK8, BMSCs were supplemented with 10% CCK8 (Dojindo, Kumamoto, Japan) in low-glucose Dulbecco's Modified Eagle's Medium for 120 min in 5% CO₂ at 37 °C. Absorbance at 450 nm was measured using a microplate reader (ELX808; BioTek, Winooski, VT, USA).
For Calcein/PI cell viability assay, after pretreatment, live BMSCs and dead BMSCs were detected using Calcein/PI assay kit (Beyotime, Shanghai, China) in accordance with the manufacturer's protocols.
For EdU staining, following treatment, BMMs viability were investigated using an EdU kit (Beyotime, Shanghai, China) in accordance with the manufacturer's protocols.

Zhang W., Zhou X., Hou W., Chen E., Ye C., Chen M., Lu Q., Yu X, & Li W. (2022). Reversing the imbalance in bone homeostasis via sustained release of SIRT-1 agonist to promote bone healing under osteoporotic condition. Bioactive Materials, 19, 429-443.

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Chondroprotective Effects of Isoginkgetin

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Selenium powder, sodium borohydride, isophorone diisocyanate (IPDI), methoxypolyethylene glycol (mPEG), dibutyltin dilaurate (DBTDL), 11-bromoundecanol, methoxypolyethylene glycols, Tetrahydrofuran (THF), hydrogen peroxide (H₂O₂), Cy 5 (Aladdin, Shanghai, China). Isoginkgetin (IGK), chloroquine (CQ), (MedChemExpress, Monmouth Junction, USA). Primary antibodies against aggrecan, collagen II, SOX9, MMP3, MMP13, ADAMTS5, ATG7, Beclin1, LC3B, SQSTM1/p62 (Abcam, Cambridge, UK), primary antibodies against BCL2, BAX and cleaved caspase-3 (Cell Signalling Technology, Danvers, USA), primary antibodies against β-actin (Proteintech, Wuhan, China). Goat anti-rabbit IgG H&L (Alexa Fluor^® 488), goat anti-rabbit IgG H&L (Alexa Fluor^® 594), (Abcam, Cambridge, UK), HRP-labeled goat anti-rabbit IgG (Proteintech, Wuhan, China). Penicillin–streptomycin (Sigma-Aldrich, St. Louis, USA). Cell counting kit-8 (CCK-8), Calcein/PI assay kit, ROS assay kit, and TUNEL apoptosis assay kit (Beyotime, Shanghai, China). Phosphate-buffered saline (PBS), FITC-annexin V/PI apoptosis detection kit, fetal bovine serum (FBS), DMEM/F12 (Thermo Fisher Scientific, Waltham, USA). Nitrocellulose membranes (Pall, New York, USA). Electrochemiluminescence substrate (Meilunbio, Dalian, China). Zoletile (Virvac, Carros, France).

Yu H., Teng Y., Ge J., Yang M., Xie H., Wu T., Yan Q., Jia M., Zhu Q., Shen Y., Zhang L, & Zou J. (2023). Isoginkgetin-loaded reactive oxygen species scavenging nanoparticles ameliorate intervertebral disc degeneration via enhancing autophagy in nucleus pulposus cells. Journal of Nanobiotechnology, 21, 99.

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