Can you LIVE dead Stain fixed cells?

Amine-reactive dyes, also known as LIVE/DEAD® fixable dead cell stains, are a class of viability dyes suitable for identifying dead cells in samples that will be fixed. These dyes cross the cell membranes of dead cells, and react with free amines in the cytoplasm.

What is LIVE dead staining?

The Live Dead assay staining solution is a mixture of two fluorescent dyes that differentially label live and dead cells. The Live cell dye labels intact, viable cells green. It is membrane permeant and non-fluorescent until ubiquitous intracellular esterases remove ester groups and render the molecule fluorescent.

What is live dead cell assay?

Live/Dead assay is a very common cell staining procedure. Live cells are stained with calcein and generate green fluorescence upon the excitation of their cytoplasm. Dead cells are labeled with the ethidium homodimer dye (EthD) which binds to their DNA and fluoresces red.

Which stains we can use for staining live and dead cells?

Viability Staining A red and green dye are added to a sample; the green dye penetrates all cells (live and dead), whereas the red dye, which contains propidium iodide, only penetrates cells whose cell membranes are no longer intact (and are therefore dead).

How do you do a live dead stain?

Protocol

  1. Thaw vial of dye.
  2. Dilute LIVE/DEAD fixable dead cell stain by adding 50 µL DMSO to vial.
  3. Add 1 mL of cells to a flow cytometer tube in protein-free buffer.
  4. Add 1 µL of diluted stain to cells.
  5. Mix cells and stain.
  6. Incubate 30 minutes.
  7. Wash cells.
  8. Analyze on flow cytometer.

Why do dead cells stain?

Fixable Dead Cell Stains Often referred to as amine-reactive dyes, these stains are based on the reaction of a fluorescent reactive dye with cellular proteins [7–10]. In live cells, only surface proteins bind to the reactive dye, resulting in dim fluorescence.

How long does live dead stain last?

These flow cytometry–based kits provide you with tools that are: Flexible—14 different LIVE/DEAD dyes excited from UV, 405, 488, 532, 561, 633, or 808 nm lasers and emission choices to different channels. Robust—clear distinction of live and dead cells is preserved for up to 30 days after fixation.

How do you do live dead assay?

For live cells, take 1 ml of cells at 5 x 106 cells/ml. For dead cells, take 1 ml of cells at 5 x 106 cells/ml, add 20 µl of 5% saponin, mix, and let stand for 10 min. This will permeabilize the cell membranes and permit EthD-1 staining of the nuclei.

How can you tell the difference between a dead cell and a live cell?

The most common way to identify dead cells is using a cell-impermeant DNA binding dye, such as propidium iodide or a dye from the STYOX series. A healthy living cell has an intact cell membrane and will act as a barrier to the dye so it cannot enter the cell.

How can you tell the difference between dead and live bacteria?

METHODS OF DIFFERENTIATING LIVE AND DEAD BACTERIA BASED ON VIABILITY PCR USING PHENANTHRIDINE DYES

  1. PCR Combined with Ethidium Monoazide Treatment (EMA–PCR)
  2. PCR Combined with Propidium Monoazide Treatment.
  3. Combined Use of EMA and PMA in PCR Assays.
  4. PCR Assay with PEMAX.
  5. Amplicon Size for Detection of Viable Microorganisms.