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