Harvard Bioscience BTX Hybrimune User manual

Hybrimune™
Hybridoma
Production System
USER’S MANUAL
Publicaon 015-100191 REV 4.0

2
Hybrimune Hybridoma Producon System
Publicaon 015-1010191 Rev 4.0 • www.btxonline.com

Since its founding in 1983, the main focus of BTX has been in the area of applying
controlled electric elds for genec engineering applicaons. Because of this,
we quickly established a reputaon as the technological leader in the elds of
electroporaon and electrofusion. Our systems have been installed in many
presgious instutes around the globe where they are used successfully for high
eciency transfecon, transformaon and cell fusion applicaons. We oer a
variety of waveforms, electrodes and chamber opons to provide you with the
best tools to achieve your goals.
We are vested in your success. To that end, the BTX technical support team
constantly tracks published literature for any reference to electroporaon and
electrofusion. We extract the pernent experimental condions and yields
from these papers to help us in our eorts to help you. In addion to tracking
publicaons, we are available to you for support at any me for advice in
experimental design, product recommendaons, troubleshoong, and any other
relevant technical advice.
We thank you again for your investment and we look forward to assisng you in
any way we can.
Finally, please read this manual carefully before aempng to operate the
electroporaon system. If you have any quesons about the unit or about
parcular applicaons, please contact us:
BTX
84 October Hill Road
Holliston, MA 01746 USA
Toll Free: 1-800-272-2775
Internaonal Callers: 508-893-8999
Fax: [phone]
Web: www.btxonline.com
Email: [email protected]
For any customers outside the US or Canada, please call your local BTX dealer or
call us directly.
A Message from BTX

3
Hybrimune Hybridoma Producon System
Publicaon 015-1010191 Rev 4.0 • www.btxonline.com
SUBJECT PAGE #
 6
 7
 8–18
Introducon 8
The Hybrimune™ Waveform Generator 8
The Large-Volume Fusion Chamber 9
Cytofusion® Medium 9
Seng up the Hybrimune™ System 10
Waveform Generator Front Panel Display 10
System Status Group 10
Process Status Group 11
Waveform Generator Interface Soware Installaon 11
Set-up using a USB-to-Serial Converter 11
System Test 12–13
Elementary Vericaon Tests 14
The Waveform Generator Interface Soware 14
The Tool Bar 15
Tools Area 15
Status Area 15–16
Last Protocol Log Window 16
Control Panel Area 16–17
Running a Protocol 17
File Management 17
Programming Waveform Protocols 18
Programming the Protocol 18
 19–26
Cell Preparaon Before Electrofusion Day 19
Myeloma Cells 19
Available Myelomas or Heteromyelomas 20
Human 20
Mouse 20
Rat 20
Rabbit 20
B-Cells 20
Immunizaon 20
Screening 20
Harvesng Spleen Cells 21
In Vitro Acvaon 21
Cell Preparaon on Electrofusion Day 21
Medium 21–22
Chamber Cleaning 22
Washes and Other Preparatory Needs 22
Cell Preparaon Process 22
Table of Contents

4
Hybrimune Hybridoma Producon System
Publicaon 015-1010191 Rev 4.0 • www.btxonline.com
SUBJECT PAGE #
Electrofusion 23
Pre-Fusion Pulse AC 23
Fusion Pulse 23
Post-Fusion Pulse AC 23
Cell Handling Aer Electrofusion 24
Immediate Post-Fusion Care of Cells 24
Materials Needed for Post Electrofusion Culture 24
Cloning Medium 1 24
Cloning Medium 2 24
Complete Growth Medium 24
HAT Stock (50X) 24
HT Stock (50X) 24
Method, Culture of Cell and Clone Selecon 25
Culture of Cells 25
Feeding Cells 25
Cloning Cells 25
Weaning Clones 25
Method of Analysis 25
Demonstraon Experiment to Illustrate Turbulence 26
Opmizaon 26
Chamber Cleaning 26
Hybrimune Electrofusion Protocol Summary 27–28
 29–43
Applicaons 29
Hybridoma Producon 29
Immunotherapy 30
Nuclear Transfer 30
Cell Fusion Methods 31
Electrofusion 32
Cell Alignment (Dielectrophoresis) 32–37
Waveforms for Electrofusion 37–43
Cell Alignment and Compression 38–39
AC Sine Wave Waveforms 39–41
Electrofusion Pulse 42
Post Fusion Pulse Sine Wave 42
Parameters Range of Hybrimune Waveform Generator 42
Published Waveform Protocols 43
 44–45
 46
Fuse Replacement 46
 47
 48
 49–51
Appendix A: Medarex Data 49–50
Appendix B: MEDCOAT 200 Summary Test Results 51
Table of Contents

5
Hybrimune Hybridoma Producon System
Publicaon 015-1010191 Rev 4.0 • www.btxonline.com
Table of Contents
 PAGE #
 8
 9
 9
 10
 12
 13
 29
 30
 30
 30
 32
 32
 34
 35
 36
 37
 38
 39
 40
 41
 42
 46