Feb 27, 2023
Evans blue assay to stain dead cells.
This protocol is a draft, published without a DOI.
- 1universitat konstanz
Protocol Citation: Santosh Sathe 2023. Evans blue assay to stain dead cells.. protocols.io https://protocols.io/view/evans-blue-assay-to-stain-dead-cells-cp3wvqpe
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 27, 2023
Last Modified: February 27, 2023
Protocol Integer ID: 77654
Abstract
Evans blue (Sigma, USA, catalog no: E2129) is a dye that can enter the compromised cell membranes of dead cells and stain them blue. I used this assay to differentiate dead cells in algal cultures of Chlorella and Chlamydomonas (the same protocol can be used for other green algae).
Evans blue assay to stain dead cells.
Evans blue assay to stain dead cells.
Contact: Santosh Sathe (santosh.sathe@uni-konstanz.de)
Introduction
Introduction
Evans blue (Sigma, USA, catalog no: E2129) is a dye that can enter the compromised cell membranes of dead cells and stain them blue. I used this assay to differentiate dead cells in algal cultures of Chlorella and Chlamydomonas (the same protocol can be used for other green algae).
Preparation
Preparation
Prepare 1% Evans blue (w/v in MilliQ water) by dissolving 0.1 gm Evans blue in 10 milliliter MilliQ water (I use Falcon tubes). Filter the dye through 0.2 Micron filters using a syringe and filter assembly (bigger pore size filters can also be used). I filter the dye to remove large undissolved particles of dye. The solution (cover it with aluminium foil) is stable at room temperate for several months.
Assay
Assay
Take 900 µl algal culture in 1.5 milliliter Eppendorf tube (I avoid 2 milliliter Eppendorf tubes as in these tubes the cells do not pellet properly; see below). If the algal cultures are less dense (less than a million cells/ milliliter), then a few milliliter of culture can be centrifuged, and the cell pellet combined and suspended in 900 µl culture media.
Add 100 ul Evans blue (from 1% dye suspension prepared above).
Incubate the mixture at room temperature for 20 minutes, preferably in the dark.
Rinse the cells with MilliQ water to remove excess dye. First, centrifuge the stained cells at 7000 RPM for 2 minutes. Remove about 900 µl dye with a 1 milliliter pipette (do not disturb the cell pellet). Since the dye is blue, the cell pellet cannot be seen during the first wash. Add 1 milliliter MilliQ water and centrifuge the cells. The cell pellet should be visible now as the leftover dye is no more concentrated. Repeat the above steps 2 to 3 times (remove 900 to 950 µl dye from the supernatant using a pipette, add MilliQ water, and centrifuge). Leave the cell pellet (after final wash) in a small volume (10 to 20 µl). Take a small amount of cell pellet (ca. 5 µl) and mount it on a clean glass slide. Put a coverslip, press gently so that cells are dispersed and observe the cells using a microscope. I use an oil immersion lens (100 X; Axioskop 40, Carl Zeiss, Germany). Count blue (dead) cells and estimate the dead cells (%) from the total cells.