Related Manuals for Harvard Bioscience BTX Hybrimune
Summary of Contents for Harvard Bioscience BTX Hybrimune
- Page 1 Hybrimune ™ Hybridoma Production System USER’S MANUAL Publication 015-100191 REV 4.0...
-
Page 2: Cloning Medium
A Message from BTX Thank you for investing in a Hybrimune system. Since its founding in 1983, the main focus of BTX has been in the area of applying controlled electric fields for genetic engineering applications. Because of this, we quickly established a reputation as the technological leader in the fields of electroporation and electrofusion. -
Page 3: Table Of Contents
Table of Contents SUBJECT PAGE # Warranty Information Safety Information Hybrimune™ System Components and Set-Up 8–18 Introduction The Hybrimune™ Waveform Generator The Large-Volume Fusion Chamber Cytofusion® Medium Setting up the Hybrimune™ System Waveform Generator Front Panel Display System Status Group Process Status Group Waveform Generator Interface Software Installation Set-up using a USB-to-Serial Converter... - Page 4 Table of Contents SUBJECT PAGE # Electrofusion Pre-Fusion Pulse AC Fusion Pulse Post-Fusion Pulse AC Cell Handling After Electrofusion Immediate Post-Fusion Care of Cells Materials Needed for Post Electrofusion Culture Cloning Medium 1 Cloning Medium 2 Complete Growth Medium HAT Stock (50X) HT Stock (50X) Method, Culture of Cell and Clone Selection Culture of Cells...
- Page 5 Table of Contents LIST OF FIGURES PAGE # Figure 1: Hybrimune System Figure 2: Large Fusion Chambers Figure 3: Cytofusion® Medium Figure 4: Hybrimune Cable Connections Figure 5: User Interface Application Screen Figure 6: Last Protocol Log Figure 7: Medarex Fusion Example Figure 8: Arizona Cancer Center Fusion Example Figure 9: Electrofusion Example Figure 10: Nuclear Transfer Example...
-
Page 6: Warranty Information
Warranty is void if the BTX Hybrimune system is changed in any way from its original factory design or if repairs are attempted without written authorization by BTX. -
Page 7: Safety Information
Make sure all connections are made properly and securely. Any signal wire connections to the unit must be no longer than three The BTX Hybrimune Systems are intended for laboratory use meters. only and can be used in research and development applications. -
Page 8: Hybrimune™ System Components And Set-Up
Hybrimune ™ System Components & Set-Up The Hybrimune Hybridoma Production System is the most sophisticated system available for fusing dissimilar cells. The system is available only under license directly from Cellectis. Please note the following important information about the system: 1. -
Page 9: The Large-Volume Fusion Chamber
Hybrimune ™ System Components & Set-Up Figure 2: Large-Volume Fusion Chamber Figure 3: Cytofusion Medium C The Large-Volume Fusion Chamber Cytofusion ® Low Conductivity Medium The Hybrimune Waveform Generator connects to a coaxial large-volume fusion chamber. The chamber produces the various BTX has developed a proprietary formula cell fusion medium electric fields required in the hybridoma production process. -
Page 10: Setting Up The Hybrimune™ System
Hybrimune ™ System Components & Set-Up Setting Up the Hybrimune™ System All of the cables necessary to install the equipment are included in the Hybrimune System. They are: 1. Serial Interface Cable, contained within the cable set 2. Line/Mains Power Cord, contained within the cable set 3. -
Page 11: Process Status Group
Hybrimune ™ System Components & Set-Up Waveform Generator Interface Process Status Group Software Installation The function of this group is to inform the user of state of an electrofusion protocol run. This group consists of three LED The Hybrimune waveform generator interface software must be indicators, and a multi-color bar graph. -
Page 12: System Test
Hybrimune ™ System Components & Set-Up System Test The following is a basic test of the waveform generator system using the preprogrammed protocol HYBRIMUNE Test.pro located in the default \ protocol folder. For this initial test, the Chamber Cable and Chamber should be fully connected, but it is not necessary to fill the chamber. -
Page 13: Figure 6: Last Protocol Log
Hybrimune ™ System Components & Set-Up What should happen… The protocol settings are downloaded to the waveform generator microprocessor. The green “Charging Power Supply On” LED will turn on, and the High Voltage window should display On. The internal high voltage power supply is now enabled and the reservoir capacitor is being charged. -
Page 14: Elementary Verification Tests
Hybrimune ™ System Components & Set-Up Elementary Verification Tests The Waveform Generator Interface Software There are two elementary tests that can be run to determine if the AC waveforms are operating properly: The Waveform Generator Interface conforms to standard Test 1 Microsoft Windows®... -
Page 15: The Toolbar
Hybrimune ™ System Components & Set-Up The Toolbar Tools Area Across the top-left of the main window is a list of standard The Tools area is found on the left side of the screen and contains Windows ® pull-down menus. a set of frequently used function buttons. -
Page 16: Last Protocol Log Window
Hybrimune ™ System Components & Set-Up Control Panel Area Chamber: This box displays whether or not the chamber cable is connected and ready to The Electroporation Mode Control Panel Area in the center of the receive pulses. screen is where the pulse-protocol parameters are set, edited, and OK: The chamber cable is connected;... -
Page 17: Running A Protocol
Hybrimune ™ System Components & Set-Up Running a Protocol • Group List: Displays the list of groups in the current protocol. The groups will be executed in The following function buttons are used to run the current the order displayed. There are three buttons to protocol: control the list: •... -
Page 18: Programming Waveform Protocols
Hybrimune ™ System Components & Set-Up Programming Waveform Protocols GROUP 2 Start 75 volts peak Pre-sine This section describes various methods to program waveform Stop 75 volts peak protocols. As stated previously there generally are three waveform Function Linear (Ramp k =1) components: Duration 20 seconds... -
Page 19: Producing Fusion Products Using Hybrimune
Producing Fusion Products Using Hybrimune ™ Cell Preparation Before This section describes a protocol to produce cell fusion using the Hybrimune System. This is a starting protocol; every lab Electrofusion has its own procedures which need to be considered. The cleaning procedure and process optimization techniques are also Myeloma Cells described. -
Page 20: Available Myelomas Or Heteromyelomas
Producing Fusion Products Using Hybrimune ™ Available Myelomas or Heteromyelomas Immunization Note: This list is not exhaustive. It was made from literature One immunization procedure follows. Immunize mice every 3 searches and a search of the ATCC and Coriell Institute’s web sites. to 4 weeks. -
Page 21: Harvesting Spleen Cells
Producing Fusion Products Using Hybrimune ™ Cell Preparation on Electrofusion Day Harvesting Spleen Cells • Euthanize immunized mice. Medium • Immerse mice in alcohol. BTX Cytofusion Medium must be used for the fusion. Establishing • Sterilely collect spleen and place into 10 ml cold DMEM. the conductivity and purity of this medium was part of the system design process. -
Page 22: Chamber Cleaning
Producing Fusion Products Using Hybrimune ™ Chamber Cleaning Cell Preparation Process The chamber must be clean for two reasons; to minimize the • Harvest cells. chance for contamination and to eliminate ions that increase • Optionally mix the cell populations at a ratio of 1:1 conductivity of medium placed in the chamber. -
Page 23: Electrofusion
Producing Fusion Products Using Hybrimune ™ Electrofusion Fusion Pulse The next step after aligning and compressing the cells is to apply The electrofusion process is done by applying a series of electrical one or more high voltage pulses to induce pores in adjacent waveforms. -
Page 24: Cell Handling After Electrofusion
Producing Fusion Products Using Hybrimune ™ Cell Handling After Electrofusion Materials Needed for Post Electrofusion Culture Immediate Post-Fusion Care of Cells Cloning Medium 1 After the electrofusion process, cells begin to completely fuse. Even • DMEM though the pores that are responsible for the fusion were formed in •... -
Page 25: Method, Culture Of Cell And Clone Selection
Producing Fusion Products Using Hybrimune ™ Methods of Analysis Method, Culture of Cells The most common method of analysis is to determine the number Culture of Cells of wells with clones (5 to 10,000 cells/well) that are secreting • Centrifuge cells. antibody and the number that are secreting antigen specific antibody. -
Page 26: Demonstration Experiment To Illustrate Turbulence
Producing Fusion Products Using Hybrimune ™ Demonstration Experiment to Chamber Cleaning Illustrate Turbulence Chamber cleaning is necessary to remove biological contaminants and ions. The electrofusion process is sensitive to the presence A simple demonstration can be done in the lab to show what of ions. -
Page 27: Hybrimune Electrofusion Protocol Summary
Computer Preparation 1. Turn on the computer. 2. Turn on the BTX Hybrimune system. 3. Load the “Hybrimune PulseAgile” program from the “Start” button in Windows (or shortcut from the desktop). -
Page 28: Hybrimune Electrofusion Protocol Summary
Producing Fusion Products Using Hybrimune ™ Hybrimune ™ Electrofusion Protocol Summary Staining (Optional QC Step) 1. Dilute a small aliquot of the fused cells 1:2 in complete media. 2. Allow the cells to rest 30 minutes. 3. Spin fused cells onto a slide at 350 RPM for 2 minutes using the cytospin machine method. -
Page 29: Cell Electrofusion Tutorial
Cell Electrofusion Tutorial Applications An example of E-fusion versus PEG was produced by M. Coccia, Ph.D., Platform Development Group at Medarex, Inc., Milpitas, There are three common uses of electrofusion. CA. Transgenic human antibody-producing mice were used in experiments comparing efficiencies of E-fusion to PEG fusion. Each •... -
Page 30: Immunotherapy
Cell Electrofusion Tutorial Immunotherapy Nuclear Transfer Another application is the ex vivo fusion of human dendritic Nuclear transfer is the transfer of a nucleus from one cell to the cells and human tumor cells removed from a patient to produce cytoplasm of another cell. -
Page 31: Cell Fusion Methods
Cell Electrofusion Tutorial Cell Fusion Methods Cell fusion is the formation of a single hybrid cell containing the nuclei and cytoplasm from more than one cell. It is accomplished by: • Bringing cells together so that they touch • Compressing the cells to increase contact area •... -
Page 32: Electrofusion
Cell Electrofusion Tutorial Electrofusion Uniform Field Non Uniform No Movement Field Movement The process of electrofusion has three steps: 1. Cell alignment and compression – 2. Fusion 3. Stabilization – – – The first step is to align the cells and then compress using special electric fields. - Page 33 Cell Electrofusion Tutorial The actual force applied is a complex formula, see Pohl, Chapter 4: e,s,w, r)] ∇ E [2p e medium The larger the force the faster the cells will move. Although the equation is complex much can be learned by looking at the terms in the equation.
-
Page 34: Figure 13: Clausius-Mossotti Function
Cell Electrofusion Tutorial A computer model of this formula is presented in Figure 13. K IS NOT UNDER USER CONTROL Clausius-Mossotti Function cell radius 7 um 1. 000 0. 800 0. 600 0. 400 0. 200 80 µS/cm 0. 000 200 µS/cm - 0. -
Page 35: Figure 14: Chamber Definitions
Cell Electrofusion Tutorial ∇ E As a / b gets small, the gap gets large and ∇E gets small and V is the characteristic of the chamber used must increase to compensate. As a / b approaches 1 the gap gets The character ∇... -
Page 36: Figure 15: Cell Alignment In Optimization Chamber
Cell Electrofusion Tutorial The following is an equivalent presentation: – 2 ∇ E ln [a / b] This shows the ∇E term has two distinct terms, the first is the amplitude of the AC waveform the second is the geometric configuration of the chamber. -
Page 37: Waveforms For Electrofusion
Cell Electrofusion Tutorial AC Waveform Amplitude In summary, the design of the Hybrimune System has been optimized for electrofusion and many of the variables are fixed The AC waveform amplitude and duration are the only variables by system design such as the chamber configuration and the under user control. -
Page 38: Cell Alignment And Compression
Cell Electrofusion Tutorial Cell Alignment and Compression A practical result of the stochastic nature of cell alignment is that the maximum yield is Pages 32–37 described the method of aligning cells using obtained when the percent of each cell dielectrophoresis. There were two important parameters, the population is 50% (shown above). -
Page 39: Ac Sine Wave Waveforms
Cell Electrofusion Tutorial 5. The initial alignment must be done slowly and deliberately so good alignments can be produced. However if a small force is used to align as required then compression will not occur. This still leaves the function of compression to increase the surface contact between adjacent cells to increase the probability of a successful fusion pair. -
Page 40: Figure 19: Ac Sine Wave Amplitude Functions
Cell Electrofusion Tutorial The voltage range of the Hybrimune voltage waveform generator Stop AC amplitude can be set from 5 to 75 volts peak in 5 volts-peak steps using several algorithms. These algorithms and their names are Start AC amplitude presented in Figure 19. -
Page 41: Figure 20: Ramp K Factor In Non-Linear Sine Wave Amplitude
Cell Electrofusion Tutorial Non-Linear Ramp (k) Amplitude This function is set by programming the start amplitude, the stop amplitude, the duration and the “Ramp k” factor. The “Ramp k” factor determines how fast the amplitude becomes non-linear. These functions are presented in Figure 20 for a Start Amplitude of 20 volts, a Stop Amplitude of 50 volts and duration of 10 seconds. -
Page 42: Electrofusion Pulse
Cell Electrofusion Tutorial Electrofusion Pulse Post Fusion Pulse AC Sine Wave The function of this waveform is to form pathways in the cell The objective of this function is to hold in place, the cells that membranes to allow the cytoplasm to mix and the cells to fuse. have been fused together, to allow them to mature. -
Page 43: Published Waveform Protocols
Cell Electrofusion Tutorial Published Waveform Protocols Waveforms which have been published are as follows: Parameter Medarex Appendix B Li, J (2006) PNAS Trevor, et al. (2004) Cancer Imm Immoth AC Pre-Pulse 40 to 60 volts peak 40 volts peak 40 volts peak 15 seconds 15 seconds 30 seconds... -
Page 44: Specifications
Specifications Waveform Generator Pulse In a Group Pulse Amplitude 100 to 1000 volts, in 5 volt steps Pulse Width 20 to 1000 µs, in 10 µs steps Number of Pulses 0 to 10, in steps of 1 Pulse Interval 0.125 to 10 sec, in 0.001 sec steps Pre-Pulse AC and Post-Pulse AC In a Group Start AC Amplitude 5 to 75 vpk, in 5 volt steps... - Page 45 Specifications Chamber Optimization Chamber Production Chamber Volume >2 ml >9 ml Outer ID 45.72 mm 45.72 mm Inner OD 38.10 mm 38.10 mm 3.81 mm 3.81 mm Well Height 5 mm 18 mm r1/r2 0.8333 0.8333 Overall Dimensions 13.5 cm x 7.7 cm x 2.2 cm 11 cm x 10 cm x 5 cm Weight 210 g (7 oz)
-
Page 46: Maintenance
Maintenance The Hybrimune Instrument requires no special maintenance other than keeping it clean. If the surface of the touch screen display or enclosure needs to be cleaned, use a standard (non-ammonia) glass cleaner or mild detergent with warm water and a soft, lint free paper or cloth towel. Do not apply the cleaning solution directly to the screen or enclosure, to avoid liquid running into other parts of the cabinet. -
Page 47: Ordering Information
Ordering Information Item No. Description Qty. Item No. Description Qty. Electrofusion Systems Accessories 47-0300N Hybrimune Electrofusion System 1 Ea 47-0001 BTXpress Cytofusion Medium C, 500 ml Volume 1 Ea 47-0305N Hybrimune Electrofusion Generator Only 1 Ea 47-0302 Hybrimune Chamber Cable 1 Ea Chambers 47-0301... -
Page 48: References
References Darveau, A, et al. Efficient preparation of human monoclonal antibody-secreting heterohybridomas using peripheral B lymphocytes cultured in the CD40 system. J Immunol Meth. 1993;159:139-143. Hui, SW, Stenger, DA. Electrofusion of cells: hybridoma production by electrofusion and polyethylene glycol. Methods Enzymol. 1993;220:212-227. James K, Bell GT. -
Page 49: Appendices
Appendices Appendix A: Medarex Data Hybridoma Production: Symbols used: E-Fusion vs. PEG Medarex Results The immunoglobulin heavy chain for IgG antibody (includes IgG 1-3) The data here was produced by Marco Coccia, Ph.D. in the Platform Development Group, Medarex, Inc., Milpitas, CA using the BTX One of two immunoglobulin light chains Hybrimune Commercial Electrofusion System. - Page 50 Appendices Appendix B: Medarex Data Experimental Results Experiment E-fusion PEG Fusion # Ag, # Ag, 1400 <1100 <400 Ag 1 Ag 2 Ag 3 Ag 4 Ag 5 Ag 5 Hybrimune Hybridoma Production System Publication 015-1010191 Rev 4.0 • www.btxonline.com...
-
Page 51: Appendix B: Medcoat 200 Summary Test Results
Appendices Appendix B: MEDCOAT 2000™ Data MEDCOAT 2000 ™ is a commercially available proprietary coating developed for stainless steel medical devices. The coating increases resistance to wear, prevents corrosion, and provides a smooth surface to clean. Physical Properties Biological Compatibility Wear Resistance Improved 1400% USP Class VI Certification... - Page 52 Contact Us US & Canadian Customers International Customers phone 508.893.8999 To locate a distributor 508.429.5732 near you, please visit our e-mail support@hbiosci.com website at www.btxonline.com website www.btxonline.com...
Need help?
Do you have a question about the BTX Hybrimune and is the answer not in the manual?
Questions and answers