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LIVE/DEAD® Violet Viability/Vitality Kit

互联网2013-11-13

1237

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.

实验试剂

Table 1. Contents and storage information.

Material	Amount	Storage	Stability
CellTrace™ calcein violet stain, lyophilized (Component A) *	5 vials, 25 μg each	≤–20°C Desiccate Protect from light	When stored as directed, kit components are stable for at least 6 months
Aqua-fluorescent reactive dye, lyophilized (Component B)	5 vials	≤–20°C Desiccate Protect from light	When stored as directed, kit components are stable for at least 6 months
DMSO, anhydrous (Component C)	500 μL	≤–20°C Desiccate Protect from light	When stored as directed, kit components are stable for at least 6 months

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.

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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