119-1. Calcein-AM/PI Living / Dead cell Double Staining Kit,

Product Information

Product Description/Introduction

Calcein AM is based on Calcein, and an acetyl methoxymethyl ester (AM) group is introduced. AM group, it not only masks the molecular part of calcium chelated by Calcein, but also enhances its hydrophobicity, so that Calcein AM can easily penetrate the living cell membrane. When Calcein AM enters the cell, it can be cut into Calcein by endogenous esterase. After losing the AM group, Calcein can’t easily pass through the cell membrane, so it stays in the cell. In addition, the molecular part of chelated calcium is exposed, and the Calcein probe can emit strong green fluorescence after combining with intracellular calcium ions. However, the dead cells lack esterase, which can’t hydrolyze Calcein AM into Calcein, so they can’t be labeled. PI (propidium iodide) can embed into the DNA double helix of cells to produce red fluorescence, but it can’t directly stain the cell membrane of living cells, only the dead cells whose cell membrane is damaged. Therefore, the combination of Calcein-AM and PI can stain the living cells and dead cells.

This kit is based on the above principle, optimized and debugged by our company. The living cells are labeled by Calcein-AM and the dead cells are labeled by PI for double staining, so that the level of living cells and dead cells can be analyzed.

Storage and Shipping Conditions

Ice bag transport; Store away from light at -20 °C, valid for 12 months.

Product Content

Attention: The above reaction times are a single 100 μL system detection.

Assay Protocol / Procedures

1. Calcein-AM/PI detection working solution configuration:

a) Return the low-temperature stored Calcein-AM solution, PI solution and detection buffer to room temperature;

b) According to the need to configure the detection working solution, configuration system can refer to the following table, pay attention to the different characteristics of different cells, according to the actual experiment. Adding or decreasing the concentration of the probe, the dilution ratio of most cells is between 1: 500 and 2000.

2.  Cell staining marker:

a)   According to the customer’s experimental arrangement, the cells were treated with pre-drug or other stimulation;

b)   For adherent cells, the supernatant should be collected first, and all cells should be collected after digestion with trypsin. For suspended cells, collect all cells directly;

c)  Centrifuge the collected cells at 800-1000 rpm for 3-5 min, remove the supernatant, and thoroughly wash the cells 2-3 times with PBS or other buffer to remove the residual esterase activity;

d)   Add the above Calcein AM/PI detection working solution to resuspend the cells to control the cell density at 1×10⁵~1×10⁶ /mL;

e)   Incubate in a cell incubator in the dark for 15-30 min.

3. Cell staining observation:

a)   According to the experimental requirements, fluorescence microscope, flow cytometry, microplate reader and other instruments can be used to observe and analyze the level of living cells and dead cells;

b)   The living cells labeled by Calcein-AM are green fluorescent, with EX/EM = 494/517 nm; The labeled dead cells of PI are red fluorescence, EX/EM=535/617 nm.

Note

1. Due to different cell-to-cell properties and different detection methods, the probe concentration and incubation time range described in step are for reference only and can be adjusted according to the specific situation.

2. All dyes have quenching problems, so please try to avoid light to slow down the quenching of fluorescence.

3. Calcein-AM is very sensitive to humidity, when you use it for the first time, please pack it properly according to the experimental arrangement. The Calcein AM/PI detection working solution should be prepared and used now.

4. For your health and safety, please wear laboratory clothes and gloves when operating.

For Research Use Only!

Ver. No.: V1.0-202102