Download Cellular studies Part 2

Cellular studies
Part 2
Sepideh Khoshnevis
Dot plot of control sample
Populations of interest
• Live cell
• Dead cell
• Debris
Gated regions for combined
forward and side scattering
Live + Dead cells
Dead + Debris
Data for fluorescence labeled dead
and apoptotic cells
Dead cells (PI)
populations are marked based on the
scattering data
Apoptotic cells (Yo-Pro)
Filter data
Dead cells (PI)
Green (PI) = Dead
Red (Yo-Pro) = Apoptosis
Black = Live
Apoptotic cells (Yo-Pro)
Filter data
Dead cells (PI)
Live cells
Apoptotic cells (Yo-Pro)
Filter data
Dead cells (PI)
• Dead cells
Apoptotic cells (Yo-Pro)
Filter data
Dead cells (PI)
Dead cells (PI)
• Debris
Apoptotic cells (Yo-Pro)
Apoptotic cells (Yo-Pro)
Viability calculation
absolute number of live cells
viability 
total number of cells  absolute number of debris
Experimental design using FCM
• Issues to resolve
– How many repetitions for each experiment
must be run to achieve satisfactory levels of
statistical confidence in data
• Uncertainty associated with experimentation
• Uncertainty associated with data analysis
– Data analysis
• Clustering
– Grouping the data points
Clustering
• K-means clustering
– Based on minimizing the Euclidean distances
between each data point and the mean of the
cluster it has been assigned to
Bootstrap
• Use bootstrap sampling to estimate
confidence interval
• Form bootstrap samples by sampling with
replacement
• Calculate statistic of interest for each
bootstrap sample
• Referred to as bootstrap replicate
• Find the mean and standard deviation of
the bootstrap replicate