1. Introduction to Flow Cytometry
IGC Workshop
Fundamentals of Flow Cytometry (cont.)
Rui Gardner
IGC – April 02, 2013

2. The Instrument 2

3. Optics (Emission Detectors)

4. Filters LP : Long Pass Filter > 500 BP : Band Pass Filter 530 / 60
Filtered
Blocked
LP : Long Pass Filter
> 500
Filtered
Blocked
BP : Band Pass Filter
530 / 60 4

5. Filters 5

6. Optical Layout 6

7. Detectors and Signal Processing

8. Detection PMT Photo Multiplier Tube
PMT’s collect photons that are then converted into voltage signals 8

9. Pulse
Flowing Stream
Voltage pulse
Laser 9

10. Pulse Parameters W H A H: A: W: 10

11. Height vs Area H H A 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... 11

12. Doublet DiscriminationW 2W H W 2H H A 2A 2A
FL-W
FL-A
Single cells
doublets
FL-H 12

13. Threshold Forward Scatter Threshold Voltage H Threshold Time W
Small Cells
and debris
Cells of Interest 13

14. Data Handling

15. Histograms
Cell Count
Fluorescence in a cell
Fluorescence

16. What are those dots?

17. Gating
Common Gate Shapes
Logical Gating
AND, OR, NOT 17

18. Gating Positive or Negative?
A “positive” cell or event is that which falls outside the “negative” gate.
Neg
Pos 18

19. Back Gating
Back gating a positive population can enrich the population of interest and help identify it correctly
CD4 FITC
Lymphocytes
CD4-
CD4+ 19

20. Dot vs Countour Plots
Dot Plots
Contour and Density Plots 20

21. Logarithmic or Linear? Anti-CD4-labeled antibody
Signals vary >100-fold
Use Log scale
Linear
Log
DNA-labeling dye
Signals vary 2- to 10-fold
Use Linear scale
Linear
Log 21

22. Acquisition How many cells should I acquire? Precision cv % = sd mean
x 100
Counting cells follows Poisson statistics:
cv % = 1
x 100
N is the number of cells counted
Example:
Population of interest is 1% of total population and want 5% precision
N =
1002
(cv %)2 =
10,000 25
= 400
40,000
Number of cells to be counted
in the region of interest
Number of total cells
to be counted 22

23. Analysis Software FlowJo X VenturiOne CellQuest Summit FCSExpress
Kaluza
FACSDiva 23

24. Analysis Software Commercial Analysis Software:
Open Source or Free software:
FlowJo X (TreeStar) - Site IGC
CellQuest/Pro (BD) - 3 IGC
FACSDiva (BD) - 2 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)
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) 24

25. Introduction to Flow Cytometry
IGC Workshop
Fundamentals of Flow Cytometry (end)
Rui Gardner
IGC – April 02, 2013