How to prepare samples for the Vitality Assay on the NC-3000

Background

The vitality assay provides easy determination of the level of reduced thiols, such as glutathione (GSH). During apoptosis, the levels of GSH decrease and this decrease can be easily detected with VitBright-48™.

GSH is involved in many cellular processes including quenching of free radicals, drug detoxification, cell signaling, and cell proliferation. The decrease in cellular GSH concentration is an early hallmark in the progression of cell death in response to different apoptotic stimuli in many cell types.

A decrease in cellular GSH concentration is an early hallmark in the progression of cell death in response to different apoptotic stimuli. Studies have shown a correlation between cellular GSH depletion and the progression of apoptosis. The decrease in GSH level in connection to apoptosis seems to be attributed to two mechanisms; 

  1. direct GSH oxidation promoted by radicals
  2. export of GSH through an ATP dependent plasma membrane transport system which is triggered by the initiation of apoptosis. 

When GSH is depleted, the cytosol is shifted from a reducing to an oxidizing environment, which may lead to a further depletion of GSH.

How to prepare samples for the Vitality Assay on the NC-3000

  1. If the cell line to be investigated is adherent or semi-adherent, then start by getting all cells into suspension using the preferred method of your laboratory (e.g. trypsin/EDTA treatment). Although the NucleoCounter® NC-3000™ is able to count aggregated cells, the accuracy is higher for single cell suspensions.
  2. Pipette a representative cell sample from the cell suspension into a microcentrifuge tube. Add one volume of Solution 5 into 19 volumes of the cell suspension. E.g., if the volume of the cell suspension is 190 μL then add 10 μL Solution 5 (VB-48™ ∙ PI ∙ AO). Mix by pipetting.
  3. Sample is now ready to run.
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