Measuring cell to cell-to-cell signaling in spot colonies (chimeric colony assay)

 

1) Inoculate a single freshly struck colony into 5 ml YPDA liquid and grow on wheel at 30°C overnight.

2) In morning harvest, transfer to 2 ml tubes, and wash 2x with 1ml dH20. Dilute 1:100 and determine cell concentration using a hemocytometer.

3) For chimeras mix 2 X 107 cells of the reporter strain (e.g. containing a LacZ or GFP reporter strains with the same number of cells of the signal strain. Harvest and re-suspend in 200ul of ddH20.

4) As a control that expression levels remains below saturation levels for the assay, prepare a “positive control” suspension containing 4 X 107 cells of the reporter strain in 200 ul. To account for background signal, prepare a “negative control” suspension containing 4 X 107 of a signal strain (i.e. lacking the reporter) in 200 ul.

5) Inoculate chimeric spot colonies and positive and negative control cells by spotting 0.5ul on to media (e.g. YNA-2, 6% OAc, 0.5% yeast extract, pH 7.0, 2% agar). When the reporter is a LacZ fusion, include X-gal in the media (see below).

6) Inoculate colonies by spotting 20 evenly spaced 0.5 ul spots in a single ring pattern with each spot 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.

7) After 3-6 d, expression of LacZ reporter is determined as described in the following link (http://sbsdev.umkc.edu/yeastcommunity/LacZ.html), and expression of XFP reporters is determined as described in the section entitled “Measuring GFP and/or mCherry expression in spot colonies”.

YNA-2-LacZ
            30 g potassium acetate
            2.5 g yeast extract
            ddH2O to 500 ml
            pH to 7.0 with KOH or acetic acid
                        Add 10g agar + stir bar
                        Autoclave, allow to cool just until bottle can be held without glove
                        Just before pouring plates, add 1.0ml X-gal (20 ug/ml in DMF) solution while stirring
                        Pour exactly 33 ml / plate using pipet

X-gal solution
            (make under hood and wear gloves)
            1 g X-gal
            50 ml Dimethyl Formamide
                        Store in glass bottle at -20°C, wrap bottle in foil

 

Citations

1) Piccirillo, Sarah, White, Melissa G., Murphy, Jeffrey C., Law, Douglas J., and Honigberg, Saul M., (2010) The Rim101p/Pacc Pathway and Alkaline pH Regulate Pattern Formation in Yeast Colonies. Genetics, 184(3): p. 707-716.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=20038633

2) 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