viSNE Settings (Cytobank)

A. 1. Sample selection: 2. Parameters selection:

Select all relevant
FCS files pre-gated for
live singlet events

Select all relevant phenotypic

markers for clustering
3. Event sampling:

Events can be sampled

proportionally or equally

Samples
B.

IL10KO #4
IL10KO #3
IL10KO #2
IL10KO #1
B6 #4

B6 #5
B6 #3
B6 #2
B6 #1

CD45
How consistent are
replicate samples in
shape and expression?
CD19

CD4
Parameters

CD8
How do plots differ geographically
between conditions?

CD11c

NKp46

CD64
How do expression profiles
change between conditions?
CD11b

C. Virus Infected Lung

(viSNE run #1)
Colored by: CD45
D. Virus Infected Lung
(viSNE run #2)
E. Run #1
(B6 #3 all events sampled)
Run #2
(B6 #3 all events sampled)
Run #3
(B6 #3 all events sampled)
NKp46+ Colored by: CD45
CD4+
Colored by: CD45 Colored by: CD45 Colored by: CD45
CD19+
CD45+
CD45-
CD8+
tSNE2

tSNE2

tSNE2

CD11b+, CD64- CD11b+, CD64+ tSNE1 tSNE1 tSNE1

What are the cellular phenotypes

of the various clusters? The viSNE plot can look different between runs, Sequential viSNE runs with identical
due to variations in viSNE calculations and events sampled can also result in
inter-run variation in sampled events. variable cellular distribution.
viSNE overlays can
identify altered
F. cluster geography
between conditions.
G. Cell type location will vary
Tumor Containing Lung
between experimental
CD8+ contexts (e.g. antibody
SigF+, CD11b+, CD64+
B6 #1 IL10KO #1 Colored by: CD45
CD4+
panel, tissue analyzed).
SigF-, CD11b+, CD64+

B6 #1 B6 #1 NKp46+
SigF+, CD11b+, CD64-
B6 #1
IL10KO #1
CD45+
CD19+
Islands that shift between
conditions identifies changes in CD45- Figure 2
cellular abundance and expression. Kimball et al