BD Accuri™C6 Flow Cytometer Overview and … Accuri C6 Flow Cytometer Overview Maria Dinkelmann,...
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Transcript of BD Accuri™C6 Flow Cytometer Overview and … Accuri C6 Flow Cytometer Overview Maria Dinkelmann,...
BD Accuri™ C6 Flow Cytometer Overview
Maria Dinkelmann, PhD
Senior Marketing Applications Specialist
BD Biosciences
Continuous Measurement of Intracellular
Calcium and Other Cellular Responses
Alfonso Blanco-Fernandez, PhD
Director of Flow Cytometry
University College Dublin
The BD Accuri C6 Flow Cytometer
An affordable, full-featured, easy-to-use flow cytometer
Two lasers and six detectors
The BD Accuri C6 flow cytometer
Innovations in all the major components of a flow cytometer
• Fluidics: Peristaltic pumps and pulse dampeners allow an open fluidics system,
miniaturization, and direct-volume measurement
• Optics: locked-down alignment
• Signal detection: broad dynamic range eliminates voltage adjustments
• Software: developed by “high-tech anthropologists” trained to facilitate human-
computer interactions
Fluidics
• Laminar flow fluidics
• Patented pulse dampeners
• Non-pressurized, peristaltic pump-
driven system
• Volume measurement for absolute
counts
• Minimum sample volume 50 µL if using
a 1.5-mL conical tube
• Up to 10,000 events/second
Sheath
Purge
Waste
Flow Cell Laser
Sa
mp
le
Dete
cto
r
Alignment and signal detection are optimized and locked
down at manufacture
FSC
SSC 488-nm
solid state laser
640-nm diode
laser
PMTs for
fluorescence
detection
Diodes for
scatter detection
Instrument-to-instrument variation is minimal
Eight-peak data, multiple C6 instruments manufactured over a
six-month period.
National Oceanic and Atmospheric Administration:
Great Lakes Microcystis research project – Lake Erie
The Ecosystem Centre:
Palmer Peninsula, Antarctica
A robust and portable tool for challenging environments
200
Voltage adjustment
Slide courtesy of Dr. Alfonso Blanco-Fernandez, University College Dublin
200 350
200
150
100
50
0
101 102 103 104 105 106 107.2
Pre-optimized detectors: fixed voltage system
Slide courtesy of Dr. Alfonso Blanco-Fernandez, University College Dublin
Intuitive software
Sample Grid
Cytometer
Status
Fluidics
Controls
Run Criteria
Real Time
Updates
Histograms
Dot Plots
Density Plots
Analysis and
Gating
Tools
Regions
Quadrants
Markers
Plot Statistics
Continuous measurement of intracellular calcium and
other cellular responses
Microscopy live cell imaging
20 – 30 cells!
0
0.2
0.4
0.6
0.8
1
1.2
0 20 40 60 79 99 119 139 159 179 199 219 238 258 278 298 318 338 357 377
Time (sec)
Flu
or.
/ a
.u.
BD FACS440
Stop-flow method
?
Stop-flow method
Stop-flow method
BD FACStar Plus
Time Zero System (Cytek)
Eric Chase &
Harvey Schulte
Keith Kelly
Time Zero System (Cytek)
BD FACStar
BD FACStar Plus
BD FACSVantage
Coulter EPICs
Coulter Elite
BD Accuri C6
Intensity
Nu
mb
er
Intensity
Nu
mb
er
BD Accuri C6
200 350 500 700
BD Accuri C6
200 350
200
150
100
50
0
101 102 103 104 105 106 107.2
BD Accuri C6
BD Accuri C6
Cliffs of Moher (Ireland)
200 350 500 700
200
150
100
50
0
101 102 103 104 105 106 107.2
BD Accuri C6
BD Accuri C6
Extremely important!!
Check the State of the instrument (QC)
Laser properly align and stable
Instrument clean,...
BD Accuri C6
Peristaltic Pumps
BD Accuri C6
Gel loading tips
BD Accuri C6
How? Pay attention to the movie
BD Accuri C6
48
8:
53
0/3
0: F
luo
-4
48
8:
53
0/3
0: F
luo
-4
48
8:
53
0/3
0: F
luo
-4
48
8:
53
0/4
0: F
luo
-4
48
8:
53
0/4
0: F
luo
-4
48
8:
53
0/4
0: F
luo
-4
Beckman Coulter Cyan ADP:
Accuri C6:
A23187 EtOH A23187 A23187 Thapsigargin
BD Accuri C6. Time limitations? F
luo
-3 :
530/3
0
Flu
o-3
: 530/3
0
Cells Air Ionophore
Flu
o-4
: 5
30/3
0
Air bubbles, any effect on the speed?
Control on the addition time for all my samples?
Time between the addition and the analysis?
63
8 :
67
5/2
5
Addition
Start Point Analysis
Start point
63
8 :
67
5/2
5
Flu
o-4
: 530/3
0
Ionophore EtOH EtOH EtOH
Calcium flux
Adapted from RS. Lewis, The molecular choreography of a store-operated calcium channel, Nature, 2007.
Fluo4™
Calcium flux
Adapted from RS. Lewis, The molecular choreography of a store-operated calcium channel, Nature, 2007.
Fluo4™
Thapsigargin
Calcium flux
Adapted from RS. Lewis, The molecular choreography of a store-operated calcium channel, Nature, 2007.
Fluo4™
Thapsigargin
2-APB
Calcium flux
488:
585/4
0:
Flu
o-4
488:
585/4
0:
Flu
o-4
488:
585/4
0:
Flu
o-4
488:
585/4
0:
Flu
o-4
488:
585/4
0:
Flu
o-4
488:
585/4
0:
Flu
o-4
Ionophore Ionophore Thapsigargin 2-APB Ionophore
Thapsigargin
Calcium flux
488 :
530/3
0:
Flu
o-4
Fragmented
cells
488:
530/3
0:
Flu
o-4
488:
585/4
0:
Flu
o-4
488:
585/4
0:
Flu
o-4
488:
585/4
0:
Flu
o-4
488:
585/4
0:
Flu
o-4
488:
585/4
0:
Flu
o-4
488:
585/4
0:
Flu
o-4
Ionophore Ionophore Thapsigargin 2-APB
Ionophore Thapsigargin
Calcium flux
488:
585/4
0:
Flu
o-4
Osmotic pressure?
Calcium flux
Erytrocytes lyse with dH2O
How do FSC vs SSC change because of osmotic pressure?
Beads
Erytrocytes lyse with dH2O
How do FSC vs SSC change because of osmotic pressure?
488:
585/4
0:
Flu
o-4
488:
585/4
0:
Flu
o-4
488:
585/4
0:
Flu
o-4
488:
585/4
0:
Flu
o-4
488:
585/4
0:
Flu
o-4
488:
585/4
0:
Flu
o-4
Ionophore Ionophore Thapsigargin 2-APB
Ionophore Thapsigargin
Osmotic pressure
???
Calcium flux
Some calcium profiles
488:
585/4
0:
Flu
o-4
488:
585/4
0:
Flu
o-4
488:
675L
P m
Ch
err
y
488: 585/40: Fluo-4
488: 585/40: Fluo-4
488:
675L
P m
Ch
err
y
488:
585/4
0:
Flu
o-4
488:
585/4
0:
Flu
o-4
Some calcium profiles in mCherry transfected cell lines W
T
mC
herr
y
Some calcium profiles in mCherry transfected cell lines
Drug
Drug
Some calcium profiles in mCherry transfected cell lines
Drug
Some calcium profiles in mCherry transfected cell lines
0.0 2.5 25.0 100.0 200.0
488:530/30: Yellow-Green npart
Time (min)
Nanoparticles uptake
NPs
48
8: 5
30
/30
: Y
ello
w -
Gre
en
NP
s
Nanoparticles uptake
Nanoparticles uptake
What if my sample has to be at 4ºC?
Nanoparticles uptake
Nanoparticles uptake
Nanoparticles uptake
Nanoparticles uptake
Fixed cell in suspension
covered by nanoparticles,
after incubation at 4°C
Nanoparticles uptake
Nanoparticles uptake
Algae physiological analysis
Kinetics of 4 different algae (FCS Express v.4)
Addition time point (2:00 min)
Kinetics of 4 different algae (FCS Express v.4)
Algae physiological analysis
Algae physiological analysis
Illumination?
Algae physiological analysis
Algae physiological analysis
Conclusions:
We have been able to establish a good
method for a continuous measurement of
morphological and intracellular calcium
changes and other cellular responses
Gethin McBean
Alice Vine
Monica Gorin
Kristina Gegenbauer
Nadezhda Glezeva
Anna Lesniak
Liam Brennan
Acknowledge:
Bob Penney
Clare Rogers
Jack Ball
Kate Easten
Grant Howes
Leo J. Ostruszka
Stuart Bradley…
Acknowledge: