What is Flow Cytometry? Flow Cytometry uic April 05, 2013 Cell Sorting Flow Cytometry Workshop IGC.
Fundamentals of Flow Cytometry (cont.) IGC – April 02, 2013 Introduction to Flow Cytometry IGC...
-
Upload
gideon-weathers -
Category
Documents
-
view
229 -
download
1
Transcript of Fundamentals of Flow Cytometry (cont.) IGC – April 02, 2013 Introduction to Flow Cytometry IGC...
Fundamentals of Flow Cytometry (cont.)
Rui GardnerIGC – April 02, 2013
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
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
17
Common Gate Shapes Logical Gating
AND, OR, NOT
Gating
18
A “positive” cell or event is that which falls outside the “negative” gate.
Positive or Negative?
PosNeg
Back Gating
19
Back gating a positive population can enrich the population of interest and help identify it correctly
CD4 FITC
Lymphocytes
CD4-
CD4+
Dot vs Countour Plots
20
Dot Plots Contour and Density Plots
Logarithmic or Linear?
21
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
Acquisition
22
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
Analysis Software
23
VenturiOne
FACSDiva
CellQuest
Kaluza
Summit
FlowJo X
FCSExpress
Analysis Software
24
Commercial Analysis Software:
FlowJo X (TreeStar) - Site License @ IGC
CellQuest/Pro (BD) - 3 Licenses @ IGC
FACSDiva (BD) - 2 Licenses @ IGC
VenturiOne (Applied Cytometry)
FCS Express (De Novo Software)
GemStone (Verity Software)
WinList (Verity Software)
ModFit LT (Verity Software)
FlowLogic (Inivai)
Kaluza (Beckman Coulter)
Guava Software (Merck-Millipore)
Open Source or Free software:
Summit (Beckman Coulter)
FlowCore (R/Bioconductor)
Weasel
Flowing Software
Attune Software (Life Technologies)
FCS Express (De Novo Software)
Cyflogic
Plate Analyzer Softwares
Plate Analyzer (Free – Purdue University)
Hyperview (Commercial – Intellicyt)
FACSDiva (Commercial – BD)
Fundamentals of Flow Cytometry (end)
Rui GardnerIGC – April 02, 2013
Introduction to Flow Cytometry IGC Workshop