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

The Instrument 2

Optics (Emission Detectors)

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

Filters 5

Optical Layout 6

Detectors and Signal Processing

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

Pulse Flowing Stream Voltage pulse Laser 9

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

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

Doublet Discrimination W 2W H W 2H H A 2A 2A FL-W FL-A Single cells doublets FL-H 12

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

Data Handling

Analysis Software Flowjo VenturiOne CellQuest Summit FCSExpress Kaluza FACSDiva 15

Histograms Cell Count Fluorescence in a cell Fluorescence

What are those dots?

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

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

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

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

Dot vs Countour Plots Dot Plots Contour Plots 22

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

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