Download Slide 1

What is Flow Cytometry?
Introduction to Flow Cytometry
IGC Workshop
Flow Cytometry
uic
Cell Sorting
IGC – Nov 12, 2009
Outline
• Cell Sorters
•
•
•
•
•
Drop Formation, Charging, and Deflection.
Drop Delay.
Sort-Drop Decisions
Purity and Recovery
Sample preparation
• Applications
Cell Sorters
FACSAria
•
•
•
•
•
MoFlo
• 100,000 events per second
• Blue, Red, UV, Yellow, Green lasers
(only 3 at the time)
• 8 simultaneous colors
• Stream in air
• Lasers aligned every time machine is
switched on
100,000 events per second
Blue, Red, Violet lasers
9 simultaneous colors
Cuvette flow cell
Fixed Alignment
Flow Sorting
Process that allows the physical separation of a cell or particle of interest
from a heterogeneous population
Droplet sorting:
 Droplet generation - Break the stream into droplets
 Charging the drop that contain the cell of interest
 Defleting the charged drop
A cell sorter sorts drops, not cells!
Drop Formation, Charging, and Deflection
• First you need to know what you want to sort
Flowcell
Charge
Collection Tubes
Aspirator
Tube
If cell of interest is within the gate at time of laser
interrogation sorter waits until cell reaches the last drop
(breakoff point) and charges the drop.
 Sheath fluid must be a conductive ionic solution
Drop Delay
At the moment of cells analysis (interrogation
point) the cell is not in a drop
All decisions are made on a theoretical
drop at the interrogation point.
Flow Cell
cuvette
Nozzle
Stream is subject to a vibration with a certain frequency
Drop
Delay
Formation of drops stabilized
Drop
Breakoff
Distan between the interrogation point and the breakoff
point constant
From the drop delay information the system can calculate the
position where the cell will be at the moment of droplet breakoff
Expectation of Sorting
The aim is:
 Sort ALL the cells of interest in the sample without losing one - Recovery
 Sort ONLY the cells we want without sorting the wrong ones – Purity
 Sort FAST without spending too much time in front of the instrument - Speed
Reality
Flow Cell
Sorting relies on some statistical rules
for randomly distributed events:
cuvette
Nozzle
Poisson Statistics
Drop
Delay
Drop
Breakoff
These rules provide the theoretical
background for calculating the achievable
sort rates and the effectiveness of sorting
Reality
1. Number of events/sec cannot
exceed 1/4 of number of drops/sec
Flow Cell
cuvette
Nozzle
1 cell for every 4 drops
(ensures that the probability of having
more than 1 cell in a drop is almost zero)
2. Cells slightly appart at the interrogation
point may end up in the same drop.
If sorting in purity mode, sorter will abort
sort decision compromising recovery.
Drop
Delay
Drop
Breakoff
Sort-Drop Decisions
A sort decision must be made for each particle.
The sort decision is based on the sort gates AND sort modes
If sorting in Purity mode:
• Decisions will compromise recovery
If sorting in Recovery mode:
• Decisions will compromise purity
If sorting in Single-Cell mode:
• High count accuracy
Purity and Recovery
Purity and recovery of the sort are mainly dependent on the
sample
1. Quality of staining
2. Concentration
3. Adhesion properties of the cells (cell-cell, cell-tube wall)
Main source of contamination during a sort are Doublets
Differently labeled cells may be attached and be deflected in the same drop.
Sample Preparation
 Cells or particles in monodispersed suspension
Suspension Cells:
 Concentration: 1 x 106 – 1x107
 Low protein concentration
Adherent Cells:
 Lower concentration
 Low protein concentration
 All sample filtered
 Collection tubes coated with FCS - Help prevent cell loss and improve cell viability
Collection Devices
Sorting can be done into:
• Tubes (15 ml, 5 ml or 1 ml)
 Up to 4 populations at the same time
• Plates (all types)
• Slides
• Petri dishes
Cell Sorting Applications
Sorting Immunophenotipic populations
CD3+
CD4+
CD25-
CD25+
CD3+ CD4+ CD25CD3+
Transcriptomics (RNA)
Genomics (DNA)
Metabolomics (metabolites)
CD3+ CD4+
CD3+ CD4+ CD25+
Fluorescence microscopy
FISH
Functional Studies
Etc.
Establishing Fluorescent Cell Lines
Carina Santos (IMM)
Interphase
Cultured
mCherry signal
Anaphase
Human hepatoma cell line
Expressing α-tubulin fused with mCherry
mCherry signal
mCherry signal
Chromosome sorting
Human cell line with translocation between
chromosome 2 and chromosome 17
AT-rich DNA signal
Normal human cell line
GC-rich DNA signal
Establishment of Cell Clones
Clone A
Clone B
Clone C
Sort single cell into each well
time
What is Flow Cytometry?
Introduction to Flow Cytometry
IGC Workshop
Flow Cytometry
uic
Cell Sorting (end)
IGC – Nov 12, 2009