Garvan Flow Cytometry Instrument talk. Given by Dr. Katharina Winnemöller, Research and Clinical Sales Manager (NSW, ACT) at the Garvan Instituteof Medical Research on the 15th October 2010. AutoMacs Pro magnetic Cell Seapartion - "the faster way to ultra-pure, viable cells"

1. MACS cell separation - the faster way to ultrapure, viable cells
+
Dr. Katharina Winnemoeller
katharinaw@miltenyibiotec.com.au

2. About us
 Germany's largest independent, privately owned biotech
company
1300 employees in 19 countries
 Cell biologists, biochemists, biophysicists, clinicians, and
engineers
all acting as a community
20% of staff in R&D, engineering & clinical development
 Manufacturing on site
minimal reliance on external suppliers

3. About us
 Founded in
Germany in 1989
 World market leader
in magnetic cell
separation (MACS)
Cologne
(Bergisch Gladbach)
corporate headquarters
 18 000+ publications
with products from
Miltenyi Biotec

4. Worldwide sales and support

5. Technology and services
 Sample preparation
 Cell Separation
 Cell Culture
 Cell Analysis
 Molecular Analysis
 Small animal imaging
 Contract MFG
 High throughput automation

6. MACS MicroBead Technology
Flexibility comes as standard

7. We are translational
 Miltenyi provides solution from bench to bedside

8. Cell Separation

9. MACS® MicroBeads – Key benefits
 MACS® MicroBeads are 50nm superparamagnetic
particles
 Flexible cell sorting strategies:
 positive selection – depletion – multiparameter sorting (no stripping!)
 Non-toxic, biodegradable:
 labeled cells go straightly to downstream experiments such as flow
cytometry, cell culture,...
 For virtually any cell type: more than 350 separation
reagents

10. MACS® Technology
Proven worldwide
– for more than 20
years
Minimal labeling
required due to column

11. Cell Separation Portfolio
390 cell separation
Stem Cells
Clinical
reagents
93
55
Immunology
Neuro
287
62
cytokine prod. cells
Whole blood
17
10
various (MHC,…)
fluorochromes
17
11
B cells
46
T cells
NK
cells
93
Granul.
13
Monos/
Mph
15
18
CSC
5
Tumor
cells
DC
31
Neural
cells
21
6
ES/iPS
cells
17
HSC
18

12. Next generation MicroBeads:
 CD4+CD25+ T reg Isolation Kit, human
 CD4+CD25+CD127dim/– Regulatory T Cell Isolation Kits
 CD4+ T Cell Isolation Kit II, human, mouse
 CD8+ T Cell Isolation Kit II, human, mouse
 Pan T Cell Isolation Kit II, human, mouse
 Plasmacytoid Dendritic Cell Isolation Kit II, human
 New Pan Monocyte Isolation Kit, human
 NK Cell Isolation Kit II, mouse
 Naive Pan T Cell Isolation Kit, human (optional TCR γ/δ T cell depletion)

13. Whole Blood MicroBead Technology
Convenience standardized
Positive cell isolation directly
from peripheral blood
Automated with the autoMACS Pro
Complete walkaway automation
Higher recovery
No RBC lysis
No density centrifugation

14. A peek into the future (mouse cells):
New Kits
Pan-DC Enrichment Kit, mouse
Pan-DC Isolation Kit, mouse
PDC Isolation Kit II, mouse
Classical DC Isolation Kit, mouse
Monocyte Isolation Kit (BM), mouse
Monocyte Isolation Kit (WB), mouse

15. Available now: fast protocols (mouse cells):

16. autoMACS Pro Separator – Key features

17. Automated multisample cell sorting
Designed and manufactured in Germany
Up to 6 samples
Option for automated labeling
Sample cooling racks
 Volumes: 0.25-50 mL (for 5, 15, 50 mL sample tubes)
 Samples stay at 4 degrees
 Choice of 12 separation programs

18. Re-usable columns
 For 14 days, or up to 100 separations
 High capacity – cell sorting within a few minutes
 105 up to 4 x 109 total nucleated cells
 up to 2 x 108 magnetically labeled cells
 up to 15 mL of whole blood

19. Numbers….
 2000 autoMACS (Pro) worldwide, 47 in Australia
 Up to 10 Mio cells per second

20. Programs and running time

21. autoMACS Pro programs
Positive selection
Possel
positive selection in standard mode for isolation of cells with normal
antigen expression and frequencies higher than 5%
4 mL/min
Possel_s
positive selection in sensitive mode for isolation of cells with low
antigen expression and frequencies higher than 5%;
1 mL/min
Posseld
positive selection in standard mode I, double-column program for
isolation of rare cells in low elution volume
4 mL/min
Posselds
positive selection in sensitive mode, double-column program for
isolation of rare cells with low antigen expression
1 mL/min
Posseld2
positive selection in standard mode II, double-column program for
isolation of rare cells
4 mL/min
Posselwb
special positive selection in double-column program for isolation
of cell subsets from whole blood
4 mL/min

22. autoMACS Pro programs
Depletion
Deplete
Depletion in standard mode for removal of cells with normal to high
antigen expression and results in better target cell yield.
4 mL/min
Depletes
depletion in sensitive mode I for removal of cells with low antigen
expression and results in better target cell purity.
1 mL/min
Depl05
depletion in sensitive mode II for removal of cells with low antigen
expression and results in stringent depletion of cells
0.5
mL/min)
Depl025
depletion in sensitive mode III for removal of cells with low antigen
expression and results in stringent depletion of cells
0.25
mL/min)
A_depl7
special depletion in standard mode for removal of cells with normal
antigen expression; used for large sample sizes where magnetically
labeled cells exceed autoMACS Column
binding capacity (2x108).
depends
A_deplS7
special depletion in sensitive mode for removal of cells with low
antigen expression; used for large sample sizes
4 mL/min

23. Results, Examples
From abundant to rare cells

24. CD4 MicroBeads, human
autoMACS™
program
Start
percentage
CD4
CD4
Purity
% CD4
cells in
negative
fraction
POSSEL
39,3%
97,9%
16,7%
2.5 min
POSSEL_S
39,3%
94,7%
3,2%
5 min
POSSELD
39,3%
99,6%
17,3%
7 min
POSSELDS
39,3%
97,2%
2,21%
8.5 min
CD4-Cy5 stained sample
Results analysed by flow cytometry
By Courtesy of A. Sheffold, A. Thiel - Berlin, Germany
time

25. Whole Blood CD14 MicroBeads, human
Anticoagulated whole blood; Purity: > 98%
program Posselwb /Rinse

26. CD34 MicroBead Kit, human
Original
sample
Human cord blood,
Purity > 95%
Program POSSELD / Rinse
Count
7-AAD
0.37%
CD34+
cells
Count
7-AAD
95.38%
CD34+
cells
CD34-PE
CD34-PE
CD34+ cells
autoMACS Separator,
by Courtesy of
Dr. Haberstock,
Dr. Allam, Hautklinik
Bonn, Germany

27. Maintenance and service

28.  Daily Maintenance
 Priming: Program Rinse (automated before first separation)
 Shut down: Program Sleep for overnight storage (fill with Ethanol)
 Periodic Maintenance
 Exchange Columns: Every 2 weeks or after 100 separations, whatever comes first
 Clean pump syringe: Every 1-2 month
 Long term storage; (substitute columns)
 Service – Service engineer and spare parts located in
Sydney

29. autoMACS Pro - the best way for automated magnetic
cell separation
Close to 400 Products for cell separation
Most flexibility
 positive or negative selection
 true Whole Blood separation, PBMC, other sources
Prettiest instrument!

30. autoMACS Pro demo – experience the difference
From November 1st you can try out the autoMACS Pro by yourself
Until November 15th
I will be around regularly to help you with your separation
Let me know what you would like to separate
 please let me know by tomorrow morning
I will order the respective samples

31. Thank you for you attention