Calcein-AM/PI Living / Dead Cell Double Staining Kit

 

Product Name	Cat. No.	Spec.
Calcein-AM/PI Living / Dead cell Double Staining Kit	G 1707-100T	100T

 

 

Calcein AM is based on Calcein (Calcein acetoxymethyl ester) and introduces an acetylmethoxymethyl ester (AM) group, which not only masks the molecular portion of Calcein that chelates calcium, but also enhances its hydrophobicity, thus allowing Calcein AM to easily penetrate the membrane of living cells. When Calcein AM enters the cell, it is able to be sheared to Calcein by endogenous cellular esterases. With the loss of the AM group, Calcein cannot easily pass through the cell membrane and is thus retained inside the cell; in addition the molecular part of the chelated calcium is partially exposed and the Calcein probe is able to emit a strong green fluorescence when bound to intracellular calcium ions. Dead cells lack esterase, which cannot hydrolyze Calcein AM into Calcein, and therefore cannot be labeled.PI (propidium iodide) is able to embed itself in the cellular DNA double helix to produce red fluorescence, but it cannot directly stain the cell membranes of living cells, and can only stain dead cells where the cell membranes have been disrupted. Thus combining Calcein-AM with PI allows for staining labeling of both live and dead cells.

This kit is based on the above principles and has been optimized and debugged by our company to perform double staining labeling by Calcein-AM labeling of live cells and PI labeling of dead cells, thus allowing analysis of both live and dead cell levels.

 

 

Ship with wet ice; Store at -20℃ away from light, valid 12 months.

 

 

Component Number	Component	G 1707-100T
G1707-1	Calcein-AM solution	20 μL
G1707-2	PI solution	20 μL
G1707-3	Detection buffer	10 mL
Manual		One copy

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

 

 

1. Preparation of Calcein AM/PI working solution

a) Remove Calcein-AM solution, PI solution and detection buffer from the freezer, then allow them to warm to room temperature.

b) Configure the working solution according to the need, the configuration system can refer to the following table, note that different cells have different characteristics, the experiment can be added or reduced according to the actual situation of the probe concentration used, most of the cell dilution ratio between 1:500-2000;

 

	10 samples	50 samples	100 samples
Calcein-AM solution	2 μL	10 μL	20 μL
PI solution	2 μL	10 μL	20 μL
Detection buffer	1 mL	5 mL	10 mL

 

2. Cell staining marker:

a) According to experimental requirement, treat the cells with drugs or other stimulations;

b) For adherent cells, collect the supernatant first, digest remaining adherent cells by trypsin and mix all cells well. For suspended cells, collect all cells directly.

c) Centrifuge the collected cells at 800-1,000 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 calcein AM/PI detection working solution to cell pellet obtained above and resuspend cells gently and thoroughly. We recommend Calcein AM/PI detection working solution to resuspend the cells to control the cell density at 1×105~1×106 /mL.

e) Incubate at 37ºC for 15-30 min in the dark.

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.

 

 

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 it can be adjusted according to the specific situation.

2. All dyes have quenching problems, Please be away from light while handling fluorescent dyes or stained samples to slow down the quenching of fluorescence.

3. Calcein-AM is very sensitive to humidity, please dispense appropriately according to the experimental arrangement when using for the first time.Calcein AM/PI assay working solution should be dispensed and used now.

4. For your health and safety, please wear safety glasses, gloves, or protective clothing.

 

For Research Use Only!