Fundamentals of Flow Cytometry (cont.)
Rui GardnerIGC – April 27, 2010
Introduction to Flow Cytometry IGC Workshop
The Instrument
2
Optics(Emission Detectors)
FilteredBlocked Blocked
BP : Band Pass Filter
530 / 60
FilteredBlocked
LP : Long Pass Filter
> 500
Filters
4
Filters
5
Optical Layout
6
Detectors and Signal Processing
Detection
8
PMT
Photo Multiplier Tube
PMT’s collect photons that are then converted into voltage signals
Pulse
9
Laser
Voltage pulse
Flowing Stream
Pulse Parameters
10
HA
W
H: A: W:
Height vs Area
11
HHA A
For non spherical cells, Height (FL-H) is not an adequate parameter to analyze
Area (FL-A) is the most adequate. However, we still need to remove doublets from the analysis...
Doublet Discrimination
12
2W
H
W
2HW
H A 2A 2A
FL-W
FL-A
Single cells
doublets
FL-H
FL-A
Single cells
doublets
Threshold
Time
Volta
ge
H
WThreshold
Forward Scatter Threshold
Small Cellsand debris Cells of Interest
13
Data Handling
Analysis Software
15
VenturiOne
FACSDiva
CellQuest
Kaluza
Summit
Flowjo
FCSExpress
100 101 102 103 104
FL1 Log
0
2393
4787
7180
9574
Counts
Histograms
Fluorescence
Cell
Coun
t
Fluorescence in a cell100 101 102 103 104
FL1 Log
0
2393
4787
7180
9574
Counts
What are those dots?
Gating
18
Common Gate Shapes Logical Gating
AND, OR, NOT
Gating
19
A “positive” cell or event is that which falls outside the “negative” gate.
Positive or Negative?
PosNeg
Back Gating
20
Back gating a positive population can enrich the population of interest and help identify it correctly
CD4 FITC
Acquisition
21
How many cells should I acquire?
Counting cells follows Poisson statistics: cv % =sd
meanx 100
N is the number of cells counted
Precision
cv % =1
x 100N
Example:
Population of interest is 1% of total population and want 5% precision
N =1002
(cv %)2=
10,00025
= 400
40,000
Number of cells to be countedin the region of interest
Number of total cellsto be counted
Dot vs Countour Plots
22
Dot Plots Contour Plots
Logarithmic or Linear?
23
Anti-CD4-labeled antibody
Linear Log
DNA-labeling dye
Linear Log
Signals vary >100-fold
Use Log scale
Signals vary 2- to 10-fold
Use Linear scale
Fundamentals of Flow Cytometry (end)
Rui GardnerIGC – April 27, 2010
Introduction to Flow Cytometry IGC Workshop
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