Prepare Stain Stock Solutions:

DAPI Stock Solution

DAPI stock solutions (10.9mM) have been prepared and are stored in the -20°C freezer. The solution lasts 6 months at 4°C once removed from the freezer.

FDA Stock Solution

FDA stock solutions are prepared by dissolving FDA (powder) in DMSO at a concentration of 1mg/mL. Care should be taken to protect the FDA from light. Stock solutions should be stored in an opaque container in the 4°C fridge for less than 6 months.

Prepare Stain for Viability Experiments:

DAPI/FDA Combined Stain

Prepare 1mL of stain immediately before use by adding 1μL of DAPI (1:1000 dilution) and 10μL of FDA (1:100 dilution) to 1mL of whole keratinocyte media.

Stain and Image Cells

Applying Stain

• After challenge/necrotic event, remove media from cells and replace with sufficient volume of prepared stain to cover all cells.
• Allow stain to remain on cells for 3 minutes
• Aspirate stain and replace with regular media, proceed immediately to imaging since cells are still alive and will provide the clearest images immediately following the staining.

Imaging Cells on Nikon 90i

• Mount the cells on the microscope and select an appropriate lens (40X recommended for viability counts)
• Focus on a field of cells in DIC, capture an image
• Without adjusting the focal plane, take an image using the FITC filter and a shutter speed of roughly 30ms
• Without adjusting the focal plane, take an image using the DAPI filter and a shutter speed of roughly 300-500ms (where nuclei clearly fall in to the category of stained or unstained)
• Merge the three images by right clicking on the small tab above the previewed images, which indicates the filter used to capture the image and select “copy”. Now right click on either of the two remaining images and paste this new layer on top of the old. Repeat for the third layer.
• NOTE: Multichannel capture achieves the same effect but has at times caused us problems with exposure time so we have used the above method instead.
• Repeat imaging steps until an adequate number of cells have been captured for counting. Do your best to sample the cells randomly in order to get an accurate count of cell death (this is why we recommend finding fields in DIC).
• Save your tri-coloured images as TIFF files for analysis later

Assess Viability

• Open saved images in NIS-elements and adjust the LUTs on your combined images until the cells are clearly separated into the live (green) or dead (blue) categories. This should be very clear when the stain works well, discard trials in which the staining is inconsistent.
• Count and record the number of alive and dead cells in each image by using the taxonomy tool in NIS. Go to the annotation tab then select the small button that has four different shapes. Clear existing data then label one indicator “Alive” and the other “Dead”. Select the appropriate shape and click on all the cells that fall into this category (NIS keeps a tally).
• Save your image after assigning cells to Alive/Dead for further review if necessary.

written by Dan Beriault, modified by Zac Robinson & Oualid Haddad December 2010