Confirm that all markers are expressed and differentiation of all cell types is occurring; replace any antibodies that do not work; revise differentiation protocol if necessary

Time course

Each day, d0–d10

Determine the peak day for expression of each marker

Hypothesis

Nanog decreases by d2. Endoderm peaks at d3-d4. Mesoderm and ectoderm peak between d6-d7

Determine a day to be used as + and – control for flow cytometry

Possible problems and solutions

Markers are expressed in undifferentiated cells— they cannot use ES cells as negative control

Determine whether markers for each cell type are expressed at similar times or have varying temporal expression

Determine the peak day for differentiation of each cell type by cycling through iterations of data collection

Time course

Peak day ± SD as determined in step 1; 24 hour intervals for iteration 1; 12 hour intervals for iteration 2

After iteration 1, the time between intervals and the time course being analyzed will be narrowed, and the number of replicates conducted will be increased

Hypothesis

Peak day for each cell type will be different; endo = d3; ecto = d7; meso = d7

Generate a comprehensive probabilistic model of ES cell differentiation over the course of time

Possible problems and solutions

Differentiation of germ layers can’t be modeled—the experiment will be conducted with late markers of differentiated cell types

Use the model generated in Step 2 as a tool to analyze the effect of STAT3 on differentiation

Time course

Peak day ± SD from the model generated in Step 2

Determine the peak day for differentiation of each cell type in cells that express dominant negative STAT3

Hypothesis

The absence of STAT3 will prevent differentiation of mesoderm and will delay or decrease ectoderm differentiation

Use statistical analysis (repeated measure ANOVA) to determine whether the loss of STAT3 leads to a change in the progression of ES cell differentiation over time

Possible problems and solutions

STAT3 has no effect on differentiation—a new target for modification of differentiation will be chosen

Confirm our results using GFP ES cell line and survival analysis