LIVE/DEAD® Violet Viability/Vitality Kit

实验概要

The LIVE/DEAD®  Violet Viability/Vitality Kit provides a two-color fluorescence cell  viability and vitality assay that is based on the simultaneous  determination of live and dead cells with two probes that measure  recognized parameters of cell health: plasma membrane integrity as a  measure of cell viability, and intracellular esterase activity as a  measure of cell vitality. Calcein violet AM and aqua-fluorescent  reactive dye are optimal dyes for this application; both dyes utilize  the violet laser allowing other laser lines to be used with more  conventional markers.

Live cells are distinguished from dead cells by the presence of  ubiquitous intracellular esterase activity, determined by the enzymatic  conversion of the virtually non-fluorescent cell-permeant calcein violet  AM to the intensely fluorescent calcein violet. In contrast, dead cells  are deficient in esterase activity and do not convert calcein AM to its  fluorescent form. However, dead cells can be positively distinguished  from live cells using the aqua-fluorescent amine-reactive dye. The  reactive dye can permeate the compromised membranes of dead cells and  react with free amines in the interior of the cell, resulting in intense  fluorescent staining of dead cells (Figures 1 and 2). In contrast, live  cells exclude the amine-reactive dye and do not exhibit strong  fluorescence.

实验原理

Principle of the method

The acetoxymethyl (AM) ester derivatives of fluorescent indicators  make up one of the most useful groups of compounds for the study of live  cells. Modification of carboxylic acids with AM ester groups results in  an uncharged molecule that can permeate cell membranes. Once inside the  cell, the lipophilic blocking groups are cleaved by nonspecific  esterases, resulting in a charged form that is retained in cells to a  much greater extent than its parent compound. The calcein violet AM  ester is colorless and non fluorescent until hydrolyzed. The polyanionic  dye calcein violet is well retained within live cells, producing an  intense uniform violet fluorescence in live cells (ex/em 400/452 nm).

Based on the reaction of a dye that is reactive with cellular  amines, dead cells may be positively distinguished from living cells.  The aqua-fluorescent reactive dye can permeate the compromised membranes  of dead cells and react with free amines both in the interior and on  the cell surface, resulting in intense fluorescent staining (ex/em  367/526 nm). In contrast, only the cell-surface amines of viable cells  are available to react with the dye, resulting in relatively dim  staining. The determinations of cell viability and cell vitality depends  on these physical and biochemical properties of the cells. As cells  die, variations in fluorescence will be observed. 
 

主要试剂

Table 1. Contents and storage information.

* Calcein violet AM may hydrolyze if exposed to moisture.

Number of labelings: At the recommended reagent concentrations and  volumes, this kit contains sufficient material to perform approximately  200 assays using flow cytometry.

Approximate fluorescence excitation/emission maxima: calcein violet 400/452 nm; aqua-fluorescent reactive dye 367/526 nm

Allow the reagents to warm to room temperature before opening the vials.

Working with the Stock Solutions

Calcein violet AM is susceptible to hydrolysis when exposed to  moisture. Once prepared, DMSO stock solutions calcein violet AM should  preferably be used within a short time period for one set of experiment,  while aqueous working solutions containing calcein violet AM should be  prepared immediately prior to use and used within one day. Once the  aqua-fluorescent reactive dye is reconstituted with DMSO, it is somewhat  unstable if exposed to moisture.

Caution

Hazards posed by these stains have not been fully investigated. DMSO  is known to facilitate entry of organic molecules into tissue. These  reagents should be handled using equipment and practices appropriate for  the hazards posed by such material. Please dispose of reagents in  compliance with all pertaining local regulations.

实验步骤

This flow cytometry protocol has been optimized using Jurkat cells (human T-cell leukemia line) at a concentration of 1 × 10⁶  cells/mL. Use of other cell types or other cell concentrations may  require optimization of staining. If another staining reaction is to be  performed on the sample, the user must determine the optimal staining  sequence for the two procedures.

1.      Allow one vial of each Component A , Component B, and Component C to come to room temperature.

2.      Prepare a 1 mL suspension of cells with 0.1–5 × 10⁶ cells/mL for each assay. Cells may be suspended in medium or buffer.

3.      Add 50 μL DMSO (Component C) to one vial aqua-fluorescent reactive dye (Component B).

4.      Add 42 μL DMSO (Component C) to one vial calcein violet AM  (Component A) to prepare a stock solution. Add 40 μL of this stock  solution to 1.25 mL of buffer or medium to make a working solution of  calcein violet AM. This working solution should be used within one day.

5.      Add 1 μL aqua-fluorescent reactive dye working solution and 5  μL calcein violet AM working solution to each mL cell suspension. Mix  the sample.

6.      Incubate the cells for 30 minutes at room temperature or on ice.

7.      Wash once and resuspend in buffer.

8.      Analyze the stained cells by flow cytometry using violet  (~405 nm) excitation and violet fluorescence emission (~450 nm) for the  calcein violet (live cells) and blue-green fluorescence emission (~525  nm) for the aqua-fluorescent reactive dye (dead cells). Minimal  compensation will be necessary.

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