[PPT] - FACSymphony Platforms for flexible research. Geoffrey Osborne PowerPoint Presentation

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FACSymphony Platforms for flexible research.

1

Geoffrey Osborne Director, Special Order Research Products

SLIDE 2

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FACSymphony™ instruments

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FACSymphony™ instruments

Overarching goal to build flexible high performance systems. Deeply imbedded in our philosophy is the drive to help our customers answer their scientific questions. Seek to do this at multiple levels:

Lasers
Detectors
Fluorophores
Software

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FACSymphony™ instruments

Deep UV Laser IR Detectors

To that end we have been developing systems with high numbers of lasers with variable laser power options.

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Flexibility presents challenges!

Overarching goal to build flexible high performance systems. Deeply embedded in our philosophy is the drive to help our customers answer their scientific questions. Seek to do this at multiple levels:

Lasers (power variability)
Detectors (sensitivity variability)
Fluorophores
Software

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Flexibility presents opportunities!

High Parameter Applications Support:

Two types of beads for system characterization are currently in development:

Those that aid in the assessment of Q and B.
Those that aid in the assessment of system function.

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New beads in development

OPV as operational voltages. (based on based

n voltage titration of CD4 stained cells, then

balanced to minimize spillover)

SLIDE 8

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FACSymphony™ Data Acquisition and Analysis challenge.

FACSChorus for FACSymphony software, currently in beta test, allows the acquisition of high parameter data past current FACSDiva limits.

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FACSymphony™ Data Acquisition and Analysis challenge.

FACSChorus for FACSymphony software acquired data showing histograms for 95 parameters. That doesn’t work for me!

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FACSymphony™ Analysis

FlowJo, LLC: Timothy Crawford, Ph.D. & Mike Stadnisky, Ph.D.

tSNE-X tSNE-Y

NK Treg CD4+ T CD8+ T B cells pDCs mDCs Monocytes

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Take home message.

BD wants to work with you in a collegial enterprise that benefits everyone involved. We recognize that there are challenges, but those are also opportunities to do things better.

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Robbie Lunt Marketing Product Manager, High Parameter

Custom Reagents Access

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Current Custom Conjugation OptiBuild Style Conjugation

Access to “Prototype” Reagents

6-8 weeks 2-3 weeks

100 200 300

Oct Nov Dec Jan Feb Mar Apr May 2016 2017

Cumulative Customs in Inventory (a.k.a. Custom Reserve) Antibodies 1750+

Legacy BVs BUVs Prototype Inventory Reagent OptiBuild Reagent

BDB_HPS_VIP@BD.com

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For internal use only

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Brent Gaylord Director R&D, Life Science Research Reagents

High Parameter Reagents

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High Parameter Color Evolution

Filling gaps Upgraded laser cores Expanded detection window: NIR New laser core: UUV Hardware Upgrades

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Roadmap Considerations

Access

BB755

New Release OptiBuild Process (2-3 weeks) BV Tandems BUV395 & BB700 Customs (6-8 weeks) BB Tandems BUV Tandems

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BD Dye Landscape

Deep UV UV Violet Blue Yellow- Green Red

FVS440
BUV615-P
BV480
BV750
BB Tandems
BB555-P (in dev)
BB755-P (new)
BYG584-P
BYG Tandems (in dev)
DUV Base (in dev)
DUV Tandems (in dev)

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Recently Launched: BB755-P

CD4 – BB755 CD8 – BB700

7th color option for the Blue Laser
Fills the gap between:

– BB700 and BB790-P1

Performance comparable to other

Brilliant Blue Tandems

BB 630 BB 660 BB 700 BB 790 BYG 750 BYG 780 Red- 750 APC- H7 Red- 805 BB750-P 7% 5% 20% 47% 2% 2% 4% 2% 2% BUV 737 BUV 805 BV 570 BV 605 BV 650 BV 711 BV 750 BV 786 BB750-P 30% 5% 13% 6% 5% 16% 53% 28%

Spillover into

Blue Violet Red UV Yel-Grn

SLIDE 19

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BSB Plus

0% 20% 40% 60% 80% 100% 120% 5 10 20 40 60 80 100 120 140 160 180 200 % of Max ul wash buffer

Decrease of +MFI with dilution

CD4 CD19

BV-BUV DDI

With the addition of reagents the increase of total volume can start to affect the overall staining with some antibodies

BSB+ is as affective as BSB with lower staining volume!

Fresh stain 1 hr cocktail 1 hr cocktail BSB 1 hr cocktail BSB+

SLIDE 20

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BD Dye Landscape

Deep UV UV Violet Blue Yellow- Green Red

FVS440
BUV615-P
BV480
BV750
BB Tandems
BB555-P (in dev)
BB755-P (new)
BYG584-P
BYG Tandems (in dev)
DUV Base (in dev)
DUV Tandems (in dev)

SLIDE 21

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UV Laser

BUV615-P FVS440

BUV615-P CD4 FVS440

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BUV615-P

7th dye choice for UV Laser
Fills gap between

– BUV563 and BUV661

Bright reagent
Manageable SO

BUV615-P CD4

BV 605 BV 650 BV 711 BUV 395 BUV 430 BUV 563 BUV 615 BUV 661 BUV 737 BUV 805 BYV 594 BYG 615 BYG 660 BYG 710

CD4-BUV615

5% 4% 3% 5% 2% 18% 32% 14% 5% 5% 41% 13% 6%

Spillover into

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FVS440

Fixable viability dye for the UV laser
Fills gap between BUV395 and BUV496 (open channel)
Compatible with common fix/perm systems
Cells may be stained, fixed, frozen, and thawed without loss of viability information

Jurkat cells treated with or without 5 µM camptothecin for 16 hours

SLIDE 24

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Violet Laser

BV750 BV480

BV480 BV510

CD4 (SK3) BV480 CD4 (SK3) BV750

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BV480

BV480 BV510

CD4 (SK3) BV480

Brighter than BV510
Less spillover into most Violet Channels

CD4

FITC BV 421 BV 605 BV 650 BV 711 BUV 470 BUV 499

BV480

3% 14% 24% 6% 1% 4% 9%

BV510

1% 7% 61% 23% 5% 3% 21%

Filters: BV421 450/40 BV480/BV510 525/50

BV480 BV510

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Using a Filter Optimized for BV421

Filter benefits for BV421

BV421 BV480 BV510 BUV395 450/40

37211 4575 928 6

431/28

36690 82 92 4 MFI in BV421 Detector From

Filter

Fluorochrome

450/50 filter works for both

BV421 and V450 reagents

431/18 filter is optimized for

BV421

Maintains 99% of the

BV421 signal.

Reduces spillover from

BV480 by 99%

Reduces spillover from

BV510 by 90%

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BV750

8th color for the Violet Laser
Option for certain IC markers

BV421 BV711 BV750 BV786 APC-Cy7 BUV737 BUV805 CD4-BV750 3% 17% 100% 76% 7% 25% 17%

BV786-A APC-A

10

2

10

3

10

4

10

5

10

2

10

3

10

4

10

5

0.09% 27.54% 0.46% 71.90%

BV786-A APC-A

10

2

10

3

10

4

10

5

10

2

10

2

10

3

10

4

10

5 20.58%

63.27% 16.01% 0.14% BV786-A APC-A

10

2

10

3

10

4

10

5

10

2

10

2

10

3

10

4

10

5 61.59%

17.83% 13.88% 6.69%

BV750 BV750 BV750

h IL-17 h IL-2 h IFN-

SLIDE 28

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Brilliant Blue Tandem Family

BB790-P BB660-P2 BB630-P BB700

BB630-P PE-CF594 BB660-P2 PE-Cy5 BB700 PerCP-Cy5.5 BB790-P PE-Cy7

As Bright as PE Tandems Compatible with PE/PE Tandems

ff Green/YG Laser

CD4 (BB660-P) CD19 (BB700)

CD45RA (PE-Cy5)

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Brilliant Blue Fluorochromes off the Blue / Yellow-Green Lasers

BB700 BB660 BB630 BB790

488nm 561nm

Minimal excitation by the 561 laser
Low spillover into equivalent Yellow-Green Detectors

– Some of the BB630 signal comes from the acceptor dye

SLIDE 30

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Recently Launched: BB755-P

CD4 – BB755-P CD8 – BB700

7th color option for the Blue Laser
Fills the gap between:

– BB700 and BB790-P1

Performance comparable to other

Brilliant Blue Tandems

BB 630 BB 660 BB 700 BB 790 BYG 750 BYG 780 Red- 750 APC- H7 Red- 805 BB750-P 7% 5% 20% 47% 2% 2% 4% 2% 2% BUV 737 BUV 805 BV 570 BV 605 BV 650 BV 711 BV 750 BV 786 BB750-P 30% 5% 13% 6% 5% 16% 53% 28%

Spillover into

Blue Violet Red UV Yel-Grn

SLIDE 31

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Yellow Green Laser

BYG Tandem Family BYG584-P BYG584-P BYG660 BYG790

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BYG584-P

Alternative to PE w/ similar brightness
Minimal Blue laser excitation
Run PE off Blue laser to get extra channel

BYG584-P PE

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Reduced Blue Laser Excitation
Resulting in lower SO/Spread issues

Brilliant YG Tandem Family

BB515 BB610 BB660 BB700 BB790 PE PE- CF594 PE- Cy5 PE- Cy7 BB515 BB610 BB660 BB700 BB790 PE PE- CF594 PE- Cy5 PE- Cy7 BB515 0.350 0.122 0.061 0.048 0.000 0.115 0.000 0.036 BB515 0.423 0.131 0.044 0.066 0.000 0.122 0.000 0.000 BB610 0.204 1.130 0.486 0.423 0.303 1.840 0.585 0.353 BB610 0.193 1.340 0.527 0.512 0.333 2.190 0.633 0.377 BB660 0.229 0.431 1.020 0.819 0.051 0.203 0.465 0.315 BB660 0.231 0.486 1.050 0.844 0.000 0.332 0.456 0.331 BB700 0.227 0.497 0.862 2.090 0.000 0.000 0.180 0.561 BB700 0.291 0.552 1.330 2.130 0.111 0.013 0.869 0.636 BB790 0.568 0.640 0.316 0.270 0.000 0.090 0.000 0.606 BB790 0.555 0.694 0.346 0.275 0.091 0.286 0.151 0.638 BYG584 0.102 0.457 0.548 0.123 0.162 2.660 0.895 0.497 PE 0.217 1.450 1.090 0.319 0.353 2.500 0.748 0.444 BYG610 0.091 0.475 0.618 0.166 0.260 0.444 1.020 0.782 PE-CF594 0.167 2.220 1.720 0.562 0.704 0.631 1.200 0.842 BYG660 0.179 0.000 1.910 0.702 0.664 0.741 1.100 1.850 PE-Cy5 0.295 0.518 5.610 2.470 2.020 0.316 0.369 2.300 BYG790 0.195 0.211 0.221 0.129 1.820 0.706 0.852 0.321 PE-Cy7 0.273 0.367 0.392 0.179 5.180 0.223 0.240 0.380

Spillover Spread Matrix (BB & BYG Reagents) Spillover Spread Matrix (BB & PE Reagents)

BYG Family PE Family

SO data courtesy of the NIH-VRC

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CD38 (BB790) CD4 (BB660) CD19 (BB700) CD197 (PE)

CD3 (BYG790) CD3 (PE-Cy7) CD45RA (BYG790) CD45RA (PE-Cy7) CD45RA (BYG660) CD45RA (PE-Cy5) CD45RA (BYG660) CD45RA (PE-Cy5)

CD197 (BYG584)

BB & BYG Tandems

CD19 (BB700)

BB & PE Tandems

CD38 (BB790) CD4 (BB660)

Brilliant YG Tandem Family

SLIDE 35

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Evolution of the Blue/YG Laser Combo

Blue Yellow- Green

FITC PerCP-Cy5.5 PE PE-CF594 PE-Cy5 PE-Cy7

Blue Yellow- Green

BB515 BB630 BB660 PE PE-CF594 PE-Cy5 PE-Cy7 BB660 BB700 BB790

Classic With BB & PE

6 10

BB515 PE BB555 BB630 BB660 BB700 BB755 BB790

With BB & BYG

Blue Yellow- Green

BYG584 BYG660* BYG720* BYG790*

12-14

High SOV Lower SOV

Colors

*Final wavelengths TBD