1. Introduction to Flow Cytometry
IGC Workshop
Fundamentals of Flow Cytometry (cont.)
Rui Gardner
IGC – April 27, 2010

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. Analysis Software Flowjo VenturiOne CellQuest Summit FCSExpress Kaluza
FACSDiva 15

16. Histograms
Cell Count
Fluorescence in a cell
Fluorescence

17. What are those dots?

18. Gating
Common Gate Shapes
Logical Gating
AND, OR, NOT 18

19. Gating Positive or Negative?
A “positive” cell or event is that which falls outside the “negative” gate.
Neg
Pos 19

20. Back Gating
Back gating a positive population can enrich the population of interest and help identify it correctly
CD4 FITC 20

21. 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 21

22. Dot vs Countour Plots
Dot Plots
Contour Plots 22

23. 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 23

24. Introduction to Flow Cytometry
IGC Workshop
Fundamentals of Flow Cytometry (end)
Rui Gardner
IGC – April 27, 2010