Measuring GFP and/or mCherry expression in spot colonies:

1) Inoculate 3 ml YPDA liquid in 15 ml tubes with freshly struck colonies from strains containing the XFP allele(s) and control strains lacking these alleles. Grow cultures on wheel at 30°C overnight.

2) Harvest, transfer to 2 ml tubes, and wash 2x with 1ml dH20. Count cell concentration with hemacytometer and resuspend a total of 4 X 107 cells in 200 ul of ddH20.

3) Inoculate colonies by spotting 0.5 ul to media. 20 evenly spaced spots are inoculated in a single ring pattern approximately 1 cm from the edge of plate. A template is placed under the plate to aid in the placement of the spots. Typically, 4-5 replicas of each strain placed on each plate, and 2-3 plates are inoculated with the same strains.

4) View under the appropriate fluorescent filter using stereomicroscope equipped with a fluorescence light source of (e.g. Olympus SZX12 microscope equipped with digital camera). Capture images and analyze data by drawing a box around the spot in Image J from pull-down menus: analyze, tools, color histogram 2 plugin (plug-in available here).

5) Record in spreadsheet program (e.g. Excel) the red (mCherry) or green (GFP) mean and standard deviation for all experimental and control spot colonies. Determine the mean of each set of 4-5 replicates for the control colony. Subtract the avg. control value from each experimental value and then calculate the mean and SEM of the experimental values.

 

Citations

1) Piccirillo, S., Morales, R., White, M. G., Smith, K., Kapros, T., Honigberg, S. M. (2015) Cell Differentiation and Spatial Organization in Yeast Colonies: Role of Cell-Wall Integrity Pathway, Genetics, 201(4): p. 1436-48.
http://www.genetics.org/content/genetics/201/4/1427.full.pdf