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

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.

Figure  1. Staining pattern of a mixture of heat-killed and untreated Jurkat  cells (human leukemia T-cell). Jurkat cells were stained according to  the protocol in the LIVE/DEAD® Violet Viability/Vitality Kit. Cells were  analyzed using a flow cytometer equipped with a 405 nm laser and a  450/50 bandpass filter for calcein violet–labeled live cells (L) and  525/50 bandpass for the aqua-fluorescent reactive dye–labeled dead cells  (D).
 Figure  2. Staining pattern of a mixture of heat-killed and untreated Chinese  hamster ovary cells (CHO cells). CHO cells were harvested using TyrpLE™  Express (Invitrogen Cat. no. 12605-010) and stained according to the  protocol in the LIVE/DEAD® Violet Viability/Vitality Kit. Cells were  analyzed using a flow cytometer equipped with a 405 nm laser and 450/50  bandpass for calcein violet–labeled live cells (L) and 525/50 bandpass  for the aqua-fluorescent reactive dye–labeled dead cells (D).

主要试剂

CellTrace™ calcein violet stain,lyophilized (Component A) *

Aqua-fluorescent reactive dye,lyophilized (Component B)

DMSO, anhydrous (Component C)

* Calcein violet AM may hydrolyze if exposed to moisture.

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.

注意事项

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.

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