Viability/Cytotoxicity Assay Kit for Live & Dead Cells (Calcein/PI) 

Calcein-AM, the acetoxymethyl ester of calcein, is highly lipophilic and cell membrane permeable. Though Calcein-AM itself is not a fluorescent molecule, the calcein generated from Calcein- AM by esterase in a viable cell emits strong green fluorescence (excitation: 490 nm, emission: 515 nm). Therefore, Calcein-AM only stains viable cells.

Propidium Iodide (PI) is a red fluorescent dye that cannot penetrate intact cell membranes. Upon cell death, PI can enter the cell and specifically bind to double-stranded DNA in the nucleus, emitting red fluorescence (Ex/Em = 535 nm/617 nm).

MCE Viability/Cytotoxicity Assay Kit for Live & Dead Cells (Calcein/PI) enables dual fluorescent staining of both live and dead cells, making it suitable for assessing cell viability and cytotoxicity. When excited at 490 nm, both live and dead cells can be observed simultaneously under a fluorescence microscope. However, when excited at 545 nm, only dead cells can be observed.

This kit is suitable for various fluorescence detection systems, including fluorescence microscopes, flow cytometers, and fluorescent microplate readers. It is compatible with the detection of most mammalian cells. However, due to the cell walls of fungi and bacteria, which hinder the entry of Calcein-AM into cells, this kit is not suitable for use with bacteria and fungi.

-20℃, 1 year

Keep away from light

1. Fluorescent dyes are subject to quenching issues. Please ensure to protect them from light to slow down fluorescence quenching.

2. Calcein-AM is prone to decomposition when moist. It is recommended to aliquot and store it at -20ºC upon first use.

3. Calcein-AM is unstable in aqueous solutions, the detection working solution must be prepared fresh and used immediately, and should not be frozen.

4. Serum and phenol red in the culture medium may affect the staining of Calcein-AM, causing an increase in fluorescence background. It is recommended to wash the cells properly before adding Calcein-AM detection working solution.

5. PI is harmful to humans, so be sure to handle it with care.

6. This product is for R&D use only, not for drug, household, or other uses.

7. For your safety and health, please wear a lab coat and disposable gloves to operate.

Since staining conditions vary depending on cell type and cell concentration, it is recommended to perform pre-experiments to determine the optimal concentrations of Calcein-AM and PI staining solutions.

1. Dead Cells preparation: Incubate cells in 70% ethanol for 30 min or in 0.1% saponin for 10 min.

Note: The method for preparing dead cells is not fixed. Alternative methods, such as treatment with 0.1%-0.5% digitonin for 10 min, can also be used.

2. Reagent Preparation: Remove the PI and Calcein-AM staining solutions to equilibrate at room temperature for 30 min.

3. PI Staining: Prepare several concentrations of PI working solutions (0.1-10 μM) using the assay buffer and stain the dead cells. Identify the concentration of PI that stains the nuclei without staining the cytoplasm.

4. Calcein-AM Staining: Prepare several concentrations of Calcein-AM working solutions (0.1-10 μM) using the assay buffer and stain dead cells. Identify the optimal Calcein-AM concentration that does not stain the cytoplasm. Subsequently, use this concentration to stain live cells and verify that live cells can be successfully stained.

Note: It is generally recommended to use the lowest possible dye concentration that provides sufficient signal intensity for the experiment.

The recommended staining concentrations for PI and Calcein-AM are 0.1–10 μM. The optimal concentration can be determined based on pre-experimental results.

The following example uses 2 μM Calcein-AM and 8 μM PI:

1. Reagent Preparation: Remove the PI and Calcein-AM staining solutions and equilibrate at room temperature for 30 min.

2. Preparation of Staining Working Solution: Add 5 μL of PI staining solution and 5 μL of Calcein-AM staining solution into 10 mL of assay buffer. Mix thoroughly to obtain the staining working solution, which can be used directly for staining.

Adherent Cells

1. Seed adherent cells into cell culture plates, microplates, or prepare cell coverslips.

Note: Suspended cells can also be prepared as cell coverslips.

2. After treating the cells according to the experimental design, wash the cells 2–3 times with PBS to completely remove residual active esterases in the culture medium.

3. Add an adequate amount of staining working solution, ensuring that the monolayer cells are fully covered.

4. Incubate at 37°C for 15 - 30 min.

Suspended Cells

1. After treating the cells according to the experimental design, centrifuge at 1,000 rpm for 3 min, discard the supernatant, and collect the cells.

Note: Recommended cell quantity is 1 × 10⁴ - 1 × 10⁵.

2. Wash the cell pellet 2 - 3 times with PBS to completely remove residual active esterases in the culture medium.

3. Resuspend the cell pellet in 100 μL of staining working solution, ensuring a cell density to 1 × 10⁵ - 1 × 10⁶ cells/mL。

4. Incubate at 37°C for 15 - 30 min.

Fluorescence Microscopy Detection

1. Adherent Cells: For cells in culture plates, aspirate the staining working solution to stop the staining. Wash the cells 2-3 times with PBS, and add a sufficient amount of assay buffer to completely cover the monolayer cells for observation. For cell coverslips, add 10 μL of assay buffer to a clean microscope slide to fully cover the coverslip for observation.

Note: Cell coverslips can be sealed with nail polish to prevent evaporation.

2. Suspension Cells: Add 10 μL of the stained cell suspension to a clean microscope slide. Seal with nail polish to prevent evaporation.

3. Detection: Use a fluorescence microscope with an excitation wavelength of 490 ± 10 nm to simultaneously observe live cells (yellow-green fluorescence) and dead cells (red fluorescence). Additionally, use an excitation wavelength of 545 nm to observe dead cells alone.

Fluorescence Microplate Reader Detection

1. Control Setup: Prepare control and experimental groups following the staining working solution preparation and staining steps.

Experimental Groups: A, B

Control Groups: Dead Cell Controls (C, D), Live Cell Controls (E, F), and Cell-Free Controls (G, H).

Note: Dead cell control can refer to the pre-experimental dead cell preparation method.

2. Adherent Cells: Can be directly detected.

3. Suspension Cells: Seed 100 μL of the stained cell suspension per well in a microplate.

Note: The minimum detectable cell count per well is 200 - 500 cells, and the maximum is 1 × 10⁶ cells.

4. Detection: Configure appropriate excitation and emission wavelengths to collect data.

For Calcein-AM: Excite at 490 ± 10 nm and collect emission signals at 530 ± 12.5 nm.

For PI: Excite at 530 ± 12.5 nm (typical Rhodamine optical filter) and collect emission signals at 645 ± 20 nm.

Note: It is recommended to use a microplate reader with optical filters to ensure signal interference is minimized.

5. Analysis and Calculation

Define the percentage of live and dead cells based on fluorescence readings.

Absolute live and dead cell counts: Generate standard curves of cell counts versus fluorescence readings (530 nm and 645 nm). Fluorescence intensity correlates linearly with cell count in the sample.

Note: Dead cells exhibit strong signals at 645 nm and weak signals at 530 nm.

Flow Cytometry Analysis

For both suspension cells and trypsin-dissociated adherent cells, follow the above staining working solution preparation and staining steps. The stained cell suspension can be directly analyzed using flow cytometry.

1. Fluorescent dyes are subject to quenching issues. Please ensure to protect them from light to slow down fluorescence quenching.

2. Calcein-AM is prone to decomposition when moist. It is recommended to aliquot and store it at -20ºC upon first use.

3. Calcein-AM is unstable in aqueous solutions, the detection working solution must be prepared fresh and used immediately, and should not be frozen.

4. Serum and phenol red in the culture medium may affect the staining of Calcein-AM, causing an increase in fluorescence background. It is recommended to wash the cells properly before adding Calcein-AM detection working solution.

5. PI is harmful to humans, so be sure to handle it with care.

6. This product is for R&D use only, not for drug, household, or other uses.

7. For your safety and health, please wear a lab coat and disposable gloves to operate.