Calcein-AM/PI Cell Viability/Cytotoxicity Assay Kit£¨Live/Dead Cell Staining Kit£©,IC-6500

Product Introduction

The Calcein-AM/PI Cell Viability and Cytotoxicity Assay Kit provides a convenient and rapid dual-fluorescence staining method for distinguishing live and dead animal cells. The assay utilizes Calcein-AM and Propidium Iodide (PI) to simultaneously monitor intracellular esterase activity and plasma membrane integrity, key indicators of cell health.

- Calcein-AM stains live cells, producing a green fluorescence.

- Propidium Iodide (PI) stains dead cells, producing a red fluorescence.

The staining procedure takes approximately 30 minutes, after which samples can be analyzed using fluorescence microscopy, flow cytometry, or a fluorescence microplate reader.

Principle:  

Calcein-AM, a cell-permeant non-fluorescent probe, is hydrolyzed by intracellular esterases in live cells to yield Calcein, a green-fluorescent compound that is retained within cells with intact membranes. Dead cells, which lack significant esterase activity or have compromised membranes, show little or no green fluorescence. PI is a red-fluorescent nucleic acid dye that can only penetrate cells with damaged membranes, staining dead cells. The distinct excitation/emission maxima of Calcein (Ex/Em = 494/517 nm) and PI (Ex/Em = 535/617 nm) allow for simultaneous detection.

Product Features

- Dual Staining: Simultaneously labels live (green) and dead (red) cells.

- Rapid & Simple: Results within 30 minutes.

- Versatile Detection: Compatible with fluorescence microscopy, flow cytometry, and microplate readers.

- High Sensitivity: Optimized for low background and clear signal differentiation.

- Convenient Format: Sufficient for 100 tests in a 96-well plate (100 µL/well at recommended dilutions).

Protocol (For reference only)

1. Preparation of Calcein-AM/PI Working Solution

Prepare the working solution immediately before use. Do not store frozen.Mix well.  

Note: The dilution factor for Calcein-AM and PI can be adjusted between 500X and 2000X depending on cell type and staining results.

2. Fluorescence Microscopy

-Seed cells in a multi-well plate, dish, or on coverslips.

-Wash cells once with PBS. For suspension cells, centrifuge at 250¨C1000 ¡Á g for 5 min and resuspend in PBS.

- Add Calcein-AM/PI working solution (100 µL/well for 96-well plate). Incubate at 37¡ãC for 30 min in the dark.

- Observe under a fluorescence microscope:  

   - Live cells: Green (Ex/Em ¡Ö 494/517 nm)  

   - Dead cells: Red (Ex/Em ¡Ö 535/617 nm)

3. Flow Cytometry

-Harvest and wash cells with PBS. Use ~1¡Á10⁶ cells per sample.

-Resuspend cell pellet in 1 mL Calcein-AM/PI working solution. Incubate at 37¡ãC for 30 min in the dark.

-Analyze directly or centrifuge and resuspend in 0.5 mL buffer.  

   - Include unstained cells (negative control) and single-stained cells (compensation controls).

-Analyze using flow cytometry with appropriate filters.

4. Fluorescence Microplate Reader (Viability & Cytotoxicity)

- Seed cells in a black 96-well plate (2,000¨C5,000 cells/well).

-Wash cells with PBS.

-Add 100 µL working solution per well. Incubate at 37¡ãC for 30 min in the dark.

-Measure fluorescence:  

   - Calcein: Ex/Em = 494/517 nm  

   - PI: Ex/Em = 535/617 nm  

-Compare RFU (Relative Fluorescence Units) between treated and control groups to assess viability/cytotoxicity.

5. Microplate Reader (Live/Dead Ratio Calculation)

-Prepare additional single-dye working solutions (Calcein-AM only, PI only).

-Set up sample and control groups as below:

-Calculate live and dead cell ratios using the formula:  

   Live Cell Ratio = ¡¾£¨R_S1-R₀1£©-£¨R_L1-R₀1£©¡¿/¡¾£¨R_L2-R₀2£©-£¨R_L1-R₀1£©¡¿

   Dead Cell Ratio =¡¾£¨R_S1-R₀1£©-£¨R_D1-R₀1£©¡¿/¡¾£¨R_D2-R₀2£©-£¨R_D1-R₀1£©¡¿

Note: For absolute cell counts, generate standard curves using known numbers of live and dead cells.

Precautions

1. Protect all reagents from light to prevent photobleaching.

2. Upon first use, aliquot Calcein-AM (1000X) and store at -20¡ãC in a sealed container.

3. Prepare Calcein-AM/PI working solution immediately before use. Do not freeze.

4. Serum and phenol red in culture medium may increase background fluorescence. Wash cells with PBS before staining.

5. For flow cytometry, adjust dye concentration as needed for optimal staining.

6. Wear lab coats and disposable gloves during use.

Frequently Asked Questions

Q1: Can I store the prepared Calcein-AM/PI working solution?  

A: No, the working solution must be used immediately and cannot be stored.

Q2: Why is background fluorescence high?  

A: Residual serum or phenol red may cause high background. Ensure thorough washing with PBS before staining.

Q3: Can I use this kit for other cell types?  

A: Yes, but optimal dye concentration and incubation time may vary. Test and optimize for each cell type.

Q4: How should I set up controls for flow cytometry?  

A: Include unstained cells, Calcein-AM-only stained cells, and PI-only stained cells for compensation and gating.

Q5: What is the maximum number of cells recommended per well in a 96-well plate?  

A: We recommend 2,000¨C5,000 cells per well for microplate reader assays.

Q6: Can this kit be used for in vivo or clinical diagnostics?  

A: No. This product is for research use only. Not for diagnostic, therapeutic, or food/drug applications.

Product Statement  

This product is intended for research use only. If used for the storage of valuable samples, please conduct small-scale testing prior to large-scale experiments. The product warranty is limited to the product itself and does not cover any other associated liabilities. Please be advised.

This product is for research use only and is not intended for clinical diagnosis or treatment.