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: