Calcein-AMPI Double Staining Kit for Live/Dead Cell Staining
Dual-color precise identification
One-click copy of information
Instruction manual- Product Description
- Composition
- Precautions
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I. Overview:
The Calcein‑AM/PI cell double‑staining kit contains two dyes: Calcein‑AM and Propidium Iodide (PI). This kit enables simultaneous visualization of live and dead cells in the same culture dish under a fluorescence microscope. Calcein‑AM can cross the cell membrane and, upon hydrolysis by intracellular esterases, releases the calcein molecule, which emits strong green fluorescence (Ex/Em: 495 nm/515 nm); thus, live cells are detected as exhibiting green fluorescence. In contrast, PI can penetrate compromised cell membranes into dead cells and intercalate into DNA double helices, producing red fluorescence (Ex/Em: 535 nm/617 nm); consequently, dead cells display red fluorescence. The maximum excitation wavelength of calcein, the hydrolysis product of Calcein‑AM, is 494 nm, with a maximum emission wavelength of 517 nm; for the PI–DNA complex, the maximum excitation wavelength is 535 nm, and the maximum emission wavelength is 617 nm.
This kit is suitable for fluorescence detection systems such as fluorescence microscopes, flow cytometers, and fluorescence microplate readers. It can be used with most eukaryotic mammalian cells, but is not applicable to bacteria or fungi.
3. Storage Conditions:
Store at -20°C, protected from light and in a sealed container; shelf life: 1 year. Transport with ice packs.
IV. Operating Steps:
1. Preparation of Calcein AM/PI staining working solution:
a. According to the assay protocol for a 96-well plate, using 100 μL of Calcein AM/PI detection working solution per well, remove the Calcein‑AM Solution and the PI Solution and allow them to equilibrate at room temperature for 30 minutes.
b. Add 1 μL of PI Solution and 1 μL of Calcein AM Solution to 1 mL of assay buffer, vortex thoroughly to mix, and prepare the working solution. The resulting working solution can be used directly for cell staining.
Note 1: To achieve optimal results, the dilution ratios of Calcein AM (1000×) and PI (1000×) can be adjusted within the range of 500–2000, depending on the cell type and the observed staining performance.
Note 2: The prepared Calcein AM/PI assay working solution must be used up in a single application and should not be frozen.
2. Fluorescence microscopy analysis:
a. Cell seeding and culture. Seed the cells into multi-well plates such as 96-well plates, cell culture dishes, or cell culture slides, and subject them to the designated treatments according to the experimental design.
b. Washing (optional). For adherent cells, remove the culture medium and wash the cells twice with PBS; for suspension cells, centrifuge at 250–1000 × g at room temperature for 5 minutes, discard the supernatant, and wash twice with PBS. Phenol red or serum may interfere with the assay performed by this kit, so be sure to remove them thoroughly.
c. Staining. Add an appropriate volume of the assay working solution. Typically, use 100 μL per well in a 96-well plate, 250 μL per well in a 24-well plate, 500 μL per well in a 12-well plate, and 1 mL per well in a 6-well plate. Incubate at 37°C in the dark for 30 minutes.
d. Detection. After incubation, observe the staining under a fluorescence microscope (Calcein AM exhibits green fluorescence, Ex/Em = 494/517 nm; PI exhibits red fluorescence, Ex/Em = 535/617 nm).
3. Flow cytometry analysis:
a. Cell preparation: Adherent cells are detached with trypsin, resuspended in culture medium, and washed twice with PBS; suspended cells are centrifuged at 250–1000 × g at room temperature for 5 minutes, the supernatant is discarded, and the cells are washed twice with PBS. The recommended cell density for each sample is 10^6 cells.
b. Staining. To the pellet of 10^6 cells from the previous step, add 1 mL of Calcein AM/PI assay working solution and resuspend to obtain a single-cell suspension. Incubate in the dark at 37°C for 30 minutes. Note: Prepare a cell sample containing only buffer as a negative control for flow cytometry; this buffer should be identical to the buffer used to prepare the Calcein AM/PI assay working solution. Additionally, prepare two extra tubes of cell samples, adding only one dye (Calcein AM or PI) to each, for compensation adjustments in single‑color flow cytometry.
c. Detection. After incubation is complete, proceed directly to flow cytometric analysis (Calcein AM emits green fluorescence, Ex/Em = 494/517 nm; PI emits red fluorescence, Ex/Em = 535/617 nm).
4. Fluorescence microplate reader assay:
a. Prepare control samples according to the experimental requirements: a cell-free control (G, H), a live-cell control (E, F), and a dead-cell control (C, D). The dead-cell control can be prepared using the method described in Step 1. If you are measuring the relative increase in live versus dead cells, controls may not be necessary.
Sample Number Group Staining solution Excitation wavelength Emission wavelength Result Naming A Sample group CalceinAM/PI 494 nm 517 nm F(517)same B Sample group CalceinAM/PI 535 nm 617 nm F(617)same C Living Cell Group PI 535 nm 617 nm F(617)max D Living Cell Group CalceinAM 535 nm 617 nm F(617)min E Dead Cell Group PI 494 nm 517 nm F(517)min F Dead Cell Group CalceinAM 494 nm 517 nm F(517)max G Cell-free group CalceinAM/PI 494 nm 517 nm F(517)0 H Cell-free group CalceinAM/PI 535 nm 617 nm F(617)0 b. Adherent cells can be directly assayed. Suspension cells: Add 100 μL of the stained cell suspension to each well of the microtiter plate. [Note: The minimum detectable cell number per well is approximately 200–500, while the typical maximum cell count per well is around 10^6.]
c. Use a fluorescence microplate reader to collect sample data with appropriate excitation and emission filters. For optimal sensitivity, it is recommended to use a microplate reader equipped with an optical filter for signal excitation, which ensures that the signals do not interfere with one another.
d. Results Analysis and Calculations:
Dead cells are characterized by a strong fluorescence signal at 645 nm and a weak fluorescence signal at 530 nm. Before calculating the results, the background fluorescence readings F(530)0 and F(645)0 can be subtracted from all values of F(530) and F(645), respectively. Live and dead
The percentage of cells can be defined as the calculation of fluorescence readings:
Live Cells% = (F(517)sam - F(517)min) / (F(517)max - F(517)min)
Dead Cells% = (F(617)sam - F(617)min) / (F(617)max - F(617)min)
Calculation of the absolute number of live and dead cells: Standard curves are generated using cell counts and fluorescence readings (at 517 nm and 617 nm), with fluorescence intensity showing a linear positive correlation with the number of cells in the sample.
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II. Kit Components:
Kit components 500T Item number Calcein-AM Reagent (1000X) 50ul 100-116A PI Stock Solution (1000X) 50ul 100-116B Staining Buffer 50ml 100-116C -
V. Precautions:
1. Since the Calcein-AM Reagent and PI Stock Solution in this kit are present in very small quantities, they may adhere to the cap or tube walls. Please centrifuge the tubes before opening.
2. Since Calcein AM (1000X) is prone to degradation in humid conditions, it is recommended to aliquot the solution appropriately upon first use and store it sealed at −20°C. For example, aliquot 10 μL per tube, seal with sealing film, wrap in aluminum foil, place in a tightly sealed plastic bag, and store at ≤−20°C, protected from light.
3. Please use the prepared staining working solution on the same day.
4. Serum and phenol red in the culture medium may affect Calcein AM staining, leading to increased fluorescence background; therefore, it is recommended to gently wash the cells prior to adding the Calcein AM working solution.
5. PI is suspected to be carcinogenic; exercise caution before use. Wear gloves, a mask, and safety goggles during handling, and avoid skin contact or inhalation. If PI comes into accidental contact with skin, rinse immediately with plenty of water.
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