Bio-Rad PDS-1000 User Manual
Biolistic®PDS-1000/He
Particle Delivery System
Catalog Numbers
165-2257 and
165-2250LEASE
to 165-2255LEASE
For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)
Warranty and Regulatory Notices
Warranty Statement
This warranty may vary outside of the continental United States. Contact your local Bio-Rad
office for the exact terms of your warranty.
Bio-Rad Laboratories warrants that the Biolistic PDS-1000/He system (catalog numbers
165-2257 and 165-2250LEASE to 165-2255LEASE) will be free from defects in material
and workmanship, and will meet all performance specifications for the period of 1 year from
the date of shipment. This warranty covers all parts and labor.
In the event that the instrument must be returned to the factory for repair under warranty, the instrument must be packed for return in the original packaging.
Bio-Rad shall not be liable for any incidental, special, or consequential loss, damage, or
expense directly or indirectly arising from the use of the Biolistic PDS-1000/He system. BioRad makes no warranty whatsoever in regard to products or parts furnished by third parties,
such being subject to the warranty of their respective manufacturers. Service under this warranty shall be requested by contacting your nearest Bio-Rad office.
The following items are considered customer-installed consumables: fuses, microcarriers,
macrocarriers, and rupture disks. These parts are not covered by this warranty. All customerinstalled parts are warranted only to be free from defects in workmanship.
This warranty does not extend to any instruments or parts thereof that have been subject
to misuse, neglect, or accident, or that have been modified by anyone other than Bio-Rad or
that have been used in violation of Bio-Rad instructions.
The foregoing obligations are in lieu of all other obligations and liabilities including negligence
and all warranties, of merchantability, fitness for a particular purpose or otherwise, expressed or
implied in fact or by law, and state Bio-Rad’s entire and exclusive liability and buyer’s exclusive
remedy for any claims or damages in connection with the furnishing of goods or parts, their
design, suitability for use, installation, or operation. Bio-Rad will in no event be liable for
any special, incidental, or consequential damages whatsoever, and Bio-Rad’s liability under no
circumstances will exceed the contract price for the goods for which liability is claimed.
Regulatory Notices
Important: This Bio-Rad instrument is designed and certified to meet EN55011,
EN50082-1, and IEC 1010-1 requirements, which are internationally accepted electrical safety standards. Certified products are safe to use when operated in accordance with the instruction manual. This instrument should not be modified or altered in any way. Alteration of this
instrument will:
Void the manufacturer’s warranty.
Void the regulatory certifications.
Create a potential safety hazard.
Note: This equipment has been tested and found to comply with the limits for a Class A
digital device, pursuant to Part 15 of the FCC rules. These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment. This equipment generates, uses, and can radiate radio frequency energy
and, if not installed and used in accordance with the instruction manual, may cause harmful
interference to radio communications. Operation of this equipment in a residential area is
likely to cause harmful interference in which case the user will be required to correct the interference at his own expense.
Table of Contents
Section 1 Biolistic PDS-1000/He Particle Delivery System..............................................1
1.1 Particle Delivery Technology ....................................................................................1
1.2 Overview of PDS-1000/He Particle Delivery System ..............................................1
1.3 Important Safety Information.....................................................................................3
1.4 Requirements for System Operation..........................................................................3
Section 2 Product Description .....................................................................................5
2.1 Packing List ................................................................................................................5
2.2 Identification of Unit Controls and Components ....................................................10
Section 3 Installation ..................................................................................................13
3.1 Connecting the PDS-1000/He System to a Helium Source ....................................13
3.2 Connecting the PDS-1000/He to a Vacuum Source................................................15
3.3 Power Cord/Voltage Regulator................................................................................16
Section 4 Operation of the PDS-1000/He Instrument ............................................17
4.1 Preparation of System Components Prior to Bombardment...................................17
4.2 Performing a Bombardment.....................................................................................22
4.3 Removal of Residual Helium Pressure–Shut Down ...............................................30
Section 5 Selection and Adjustment of System Bombardment Parameters........30
5.1 Overview -Matrix of Variables: Cell Types, Settings and Conditions ...................30
5.2 Vacuum Level in Bombardment Chamber..............................................................31
5.3 Helium Pressure / Rupture Disk Selection ..............................................................31
5.4 Solenoid Valve Adjustment .....................................................................................32
5.5 Vacuum Flow Rate Control Valves.........................................................................32
5.6 Distance Between Rupture Disk and Macrocarrier.................................................32
5.7 Distance Between Macrocarrier and Stopping Screen............................................32
5.8 Distance Between Stopping Screen and Target Shelf
(microcarrier flight distance)....................................................................................32
5.9 Microcarrier Selection..............................................................................................33
5.10 Preparation of Biological Material for Bombardment ............................................34
Section 6 Troubleshooting......................................................................................................37
6.1 Rupture Disk Bursts at Incorrect Pressure...............................................................37
6.2 Stopping Screen Forced Through Screen Support Ring .........................................37
6.3 Excessive Gas Usage................................................................................................38
6.4 Chamber Will Not Hold Vacuum ............................................................................38
6.5 Sample Damage From Gas Pressure Wave .............................................................38
6.6 Unit Will Not Pressurize Gas Acceleration Tube....................................................39
Section 7 Product Information..................................................................................40
7.1 Biolistic System........................................................................................................40
7.2 Spare Parts ................................................................................................................41
Section 8 Appendices ..............................................................................................................43
8.1 Cleaning the PDS-1000/He Device .........................................................................43
8.2 Metal Case Version ..................................................................................................43
8.3 Specifications ...........................................................................................................46
8.4 Performing a Bombardment—Quick Guide Tear-Out............................................47
Section 1
Introduction to Particle Delivery
1.1 Particle Delivery Technology
Biolistic particle delivery is a method of transformation that uses helium pressure to introduce DNA-coated microcarriers into cells. Microprojectile bombardment can transform such
diverse targets as bacterial, fungal, insect, plant, and animal cells and intracellular organelles.
Particle delivery is a convenient method for transforming intact cells in culture since minimal
pre- or post-bombardment manipulation is necessary. In addition, this technique is much easier and faster to perform than the tedious task of micro-injection. Both stable and transient
transformation are possible with the Biolistic particle delivery system.
1.2 Overview of PDS-1000/He Particle Delivery System
The Biolistic System
The Biolistic PDS-1000/He instrument uses pressurized helium to accelerate sub-cellular sized microprojectiles coated with DNA (or other biological material) over a range of
velocities necessary to optimally transform many different cell types. The system consists of
the bombardment chamber (main unit), connective tubing for attachment to vacuum source,
and all components necessary for attachment and delivery of high pressure helium to the main
unit (helium regulator, solenoid valve, and connective tubing).
Fig. 1.1. Unit components, front view.
1
Helium
Pressure
Regulator
Vacuum (reinforced PVC)
Tygon Tubing
2.5 ft PEEK Tubing
6 ft PEEK Tubing
Tank of Pressurized Helium
(user provided)
Rupture Disk
Retaining Cap
3-Way Helium Metering
(solenoid) valve
Microcarrier Launch
Assembly
Helium Regulator
Target Plate Shelf
The Biolistic Process
The Biolistic PDS-1000/He system uses high pressure helium, released by a rupture disk,
and partial vacuum to propel a macrocarrier sheet loaded with millions of microscopic tungsten or gold microcarriers toward target cells at high velocity. The microcarriers are coated with
DNA or other biological material for transformation. The macrocarrier is halted after a short
distance by a stopping screen. The DNA-coated microcarriers continue traveling toward the
target to penetrate and transform the cells.
The launch velocity of microcarriers for each bombardment is dependent upon the helium pressure (rupture disk selection), the amount of vacuum in the bombardment chamber, the
distance from the rupture disk to the macrocarrier (A), the macrocarrier travel distance to the
stopping screen (B), and the distance between the stopping screen and target cells (C).
Fig. 1.2. The Biolistic bombardment process.
Design Improvements
The original Biolistic device used a gunpowder explosion to accelerate DNA-coated
microcarriers into target cells. The helium technology used in the current PDS-1000/He system has primary advantages of providing cleaner, safer, and more reproducible particle acceleration. This stems from the use of rupture disks that burst at a defined pressure. In addition,
helium inflicts less tissue damage. Bulletin 1689 offers a comparative analysis of gunpowder
and helium target patterns and transformation efficiencies.
The PDS-1000/He device was updated in March 1995 to improve the quality of key components. We removed parts originally designed to operate with the gunpowder acceleration
method. The most notable change was the conversion of the material used for the bombardment chamber from metal to a strong, lightweight plastic. This makes the instrument easier
to transport and clean, with no change in bombardment performance (identical internal chamber dimensions). An over-pressure relief valve and a particle filter on the vacuum vent supply were also added.
The actual steps for performing a particle bombardment of a biological sample are
unchanged, and the consumables are also the same with the plastic case version. Extensive testing involving the genetic transformation of yeast, plant, and animal cells by both Bio-Rad
and independent researchers demonstrated that the gene transfer results obtained with the
new plastic chamber design are equivalent to those of the previous metal chamber model.
See Appendix 8.2 for a description of parts unique to the metal-chamber design.
2
Before
Gas Acceleration Tube
Rupture Disk
Macrocarrier
Stopping Screen
Target Cells
DNA-coated Microcarriers
After
1.3 Important Safety Information
Pressurized Helium Safety Information
Caution: Although helium is neither toxic nor flammable, all gases under pressure are potentially dangerous if used improperly. Never use a helium tank with, or attach a tank to, the
PDS-1000/He system unless the tank is properly secured. Follow the instructions provided with
the helium cylinder from the supplier and those that are applicable for your institution (site
safety officer). Bio-Rad has supplied tubing, fittings, a control valve, and a pressure regulator capable of safely handling the high pressure helium gas used in the Biolistic bombardment process. These components have been carefully selected and are the only parts to be
used with the PDS-1000/He system.
Power Safety Information
Figure 1.3 shows the serial number certification label which is found at the rear of the
Biolistic PDS-1000/He unit. This label provides the manufacturing data about the instrument,
its voltage settings, and CDRH standards for electrical safety. This instrument and its accessories conform to the IEC and CDRH standards for electrical safety.
Fig. 1.3. Instrument serial number label on the rear of the instrument.
1.4 Requirements for System Operation
Selecting Site for Operation
Prepare a space 61 cm wide x 46 cm long x 61 cm high (24 inches x 18 inches x 24 inches)
preferably in a bio-containment hood or other tissue preparation area, near a standard electrical
outlet (110 V/60 Hz in the U.S.). Also, allow for placement of the vacuum pump near the site of
operation if house vacuum is not used (see Vacuum Supply).
User Supplied Components
Helium Supply
Only helium gas is to be used with PDS-1000/He system. The low atomic weight of helium permits maximum gas expansion into the bombardment chamber. Thus, sufficient acceleration of the DNA-coated microcarriers is generated for penetration of the target cell membrane.
Obtain a high pressure (2,400 to 2,600 psi) tank of high purity helium for optimization of
bombardment conditions for the biological system of choice. This allows use of all of the rupture
disks (the highest disk has a 2,200 psi rating). Only grade 5 (99.999%) or grade 4.5 (99.995%)
helium is to be used, since helium of a lesser grade contains contaminating material which may
obstruct gas flow within the PDS-1000/He system, as well as contaminate the biological sample.
Follow all safety instructions provided by helium supplier for helium tank installation.
3
Made in U.S.A.
Model No.
Voltage
Serial No.
The helium pressure regulator (supplied) has a CGA 580, female fitting (standard in the
United States) for attachment to the user-supplied helium tank. An adaptor to this fitting may
be required outside the United States. Contact your local Bio-Rad office for information on
the helium pressure regulator adaptor requirements in your location.
A user-supplied, 1 inch adjustable wrench is required for attachment of the regulator to
a helium tank having a capacity of 55 cubic feet or greater.
Vacuum Source
The main unit of the PDS-1000/He system must be connected to a vacuum source capable of evacuating the bombardment chamber to a minimum of 5 inches of mercury for operation. This minimum vacuum requirement is part of the instrument safety system (the chamber
door must be sealed for helium pressure to be delivered into the main unit). Tubing is supplied
to connect the PDS-1000/He system to vacuum source.
For maximum evacuation capacity, we recommend connecting the PDS-1000/He device
to an oil-filled, rotary vane vacuum pump, either single or dual stage, with an pumping speed
of 90–150 liters/minute (3–5 cubic feet/minute). This pumping rate minimizes the time target
cells are exposed to vacuum. Oil for the vacuum pump must be supplied by the user. An
exhaust mist eliminator on the vacuum pump is also recommended.
The level of the vacuum required in the bombardment chamber depends on the biological system being targeted for transformation. Higher vacuum reduces drag forces on microcarriers during helium-driven acceleration. During the brief bombardment process (less than
1 minute), typical protocols require a vacuum level within the bombardment chamber between
15–29 inches of mercury. Some cells, tissues, and intact plant cells require a high vacuum
(up to 28 inches of mercury) for efficient transformation.
House vacuum may be sufficient for bombardment of certain cell types (mammalian
cells), but house vacuum pumping rates can fluctuate and vary greatly in overall evacuation
capacity (typically 20 inches of mercury, maximum).
Consumables
The 500 Optimization Kit (catalog number 165-2278) provides the consumables needed
for 500 bombardments. It is recommended for users who have yet to determine the optimal
conditions for the bombardment of the biological system of interest. The kit contains 0.25 g
each of 0.6 µ, 1.0 µ, and 1.6 µ gold microcarriers, 100 each of the nine different rupture disks
(ranging from 450 psi to 2,200 psi), 500 macrocarriers, and 500 stopping screens. After optimal conditions are determined, Standard Pressure Kits are available (see Section 7 or the current Bio-Rad catalog for a complete listing).
Additional Laboratory Supplies and Equipment
Vortex mixer is needed for microcarrier preparation.
Common laboratory supplies, such as 95% ethanol, pipettes, etc. are required, as cited in
standard protocols.
Additional Items Available from Bio-Rad
The Yeast Optimization Kit (catalog number 170-3100) allows first-time users to become
familiar with the Biolistic instrument. It is also helpful for experienced users wishing to
periodically standardize their bombardment conditions. This kit provides all of the biological
material needed to transform yeast. Yeast provides a system that is quickly and easily assayed.
The kit demonstrates the effect of varying the DNA concentration, rupture disk pressure, and
the target distance (from the stopping screen), and includes Saccharomyces cerevisiae strain
4
948, YEp352 DNA, CaCl2, spermidine, culture medium, and plating medium. Enough material for 60 bombardments is provided.
Macrocarrier Holders, set of 5 (catalog number 165-2322), are included with the Biolistic
PDS-1000/He system. Additional holders are desirable to facilitate a series of bombardments
in one experiment.
The Disk-Vac (catalog number 165-2323) is a small pen-shaped device capable of generating a suction for efficient handling of rupture disks and macrocarriers. Use of the Disc-Vac
reduces static generated during manipulation and prevents contamination with glove powder
or oil from skin.
Customers outside the continental USA and Canada will require a Voltage Converter
(catalog number 165-2259). To use the system, you must locally obtain a cord set which has
an IEC/320/CEE 22 connector on one end. This connects to the Voltage Converter and is the
type commonly found on computers or televisions. The other end of the power cord will
terminate in a plug which will fit the receptacle used in your location. Contact your local
Bio-Rad office for more information on this Voltage Converter.
Section 2
Product Description
2.1 Packing List
Check the items received with your PDS-1000/He unit against the list below. If items are
missing, contact your local Bio-Rad office.
Instruction Manual
Pressure Regulator for Helium Cylinder (with in-line 0.45 µ filter)
Fig. 2.1. Helium pressure regulator.
5
6.0 ft PEEK plastic tubing (1/16" OD x .010" ID tubing & fittings), used to connect 3-way
helium metering (solenoid) valve to helium pressure regulator
2.5 ft PEEK plastic tubing (1/16" OD x .010" ID tubing and fittings), used to connect
3-way helium metering (solenoid) valve to rear of main unit
Fig. 2.2. 6 ft and 2. 5 ft PEEK tubing.
3-Way Helium Metering (solenoid) Valve, with attached cord for power connection to
main unit
Power cord (US and Canada only, 120 V three prong plug)
Fig. 2.3. 3-way helium metering (solenoid) valve.
6
Power Cord
To Unit
6 ft PEEK Tubing
To Helium Tank
2.5 ft PEEK
Tubing To Unit
Tools
3 Hexagonal gap setting tools (1 each- 1/8", 1/4", and 3/8"), used to set gap between the
bottom of the rupture disk retaining cap and the lid of the microcarrier launch assembly
1 3/16" hex key (Allen) wrench, used for removal of gas acceleration tube (service only)
1 1/8" hex key (Allen) wrench, used for releasing set screw in microcarrier launch assembly shelf
2 1/4" x 5/16" open-end wrenches, used for connecting small Swagelock fittings of plastic
PEEK tubing to rear of instrument, external solenoid valve, and helium pressure regulator
1 torque wrench for rupture disk retaining cap
1 seating tool for macrocarriers
Fig. 2.4. PDS-1000/He tools.
7
PDS-1000/He Main unit (bombardment chamber with control panel and gauges; shipped
fully assembled)
Reinforced PVC vacuum tubing (1/2" ID x 3/4" OD, 5 ft. length & fitting) attached to rear
of unit by clamp assembly (centering ring, vacuum hose clamping ring and nozzle adaptor; see Section 3.2 for individual components)
Rupture disk retaining cap (with torque wrench placement holes; Figure 10), attached to
gas acceleration tube within bombardment chamber
Fig. 2.5. PDS-1000/He main unit.
Fig. 2.6. Rupture disk retaining cap.
8
Microcarrier
Launch Assembly
Vacuum Tubing
Rupture Disk
Retaining Cap
Microcarrier launch assembly (shipped fully assembled) consists of the following:
Launch Assembly Shelf with Recessed Set Screw
Macrocarrier Cover Lid
Adjustable Nest
Fixed Nest with Retaining Spring
Stopping screen Support Ring
Spacer Rings, 5 mm height, 2
Macrocarrier Holders, 5, for use within microcarrier launch assembly, after macrocarrier
is inserted using macrocarrier insertion tool
Target Plate Shelf
Fig. 2.7. Microcarrier launch assembly (A) and disassembled components (B).
Fig. 2.8. Target plate shelf.
9
A
B
2.2 Identification of Unit Controls and Components
The following is a brief description of the operation controls for the PDS-1000/He unit
(Figure 2.9)
Table 2.1. Unit Controls and Components
Controls and
Components Description
Front View- Exterior
Power Switch, ON/OFF Controls supply of line electrical power to the
instrument.
Fire Switch Controls flow of helium into Gas Acceleration Tube by
activating Solenoid Valve. Illuminated red when
enabled, i.e. when safety interlock is satisfied that at
least 5 inches Hg. vacuum is present in chamber.
Fire Switch must be held ON continuously until
Rupture Disk bursts; then release Fire Switch to stop
flow of helium.
If the Fire Switch is released before the disk ruptures,
the helium is vented via a safety vent in the external
three-way metering (solenoid) valve.
Vac/Vent/Hold Switch Controls application of vacuum to bombardment
chamber. Vac applies vacuum from line source. Vent
releases vacuum using filtered air. Hold maintains
vacuum by isolating chamber.
Bombardments should be performed with this switch
in “Hold” position.
Vacuum Gauge Indicates level of vacuum in bombardment chamber,
in inches of mercury where zero equals ambient atmospheric pressure.
Vacuum/Vent Rate Regulate rate of application and relief of vacuum in
Control Valves bombardment chamber. Clockwise rotation closes valves.
Helium Pressure Gauge Indicates helium pressure in Gas Acceleration Tube,
in psi. When solenoid valve is activated by Fire Switch,
the needle in this oil-filled gauge rotates clockwise until
rupture disk bursts. Watch this gauge carefully during a
bombardment and note the actual rupture pressure.
Gas Acceleration Tube Helium accumulates within this tube when it is sealed
by rupture disk at chamber-end of Tube. Helium PEEK
tubing connects at top of Gas Acceleration Tube outside,
rear of chamber.
Bombardment Chamber Holds Rupture Disk, Microcarrier Launch Assembly, and
biological target under vacuum during a bombardment.
10
Fig. 2.9. Front view of PDS-1000/He unit.
Table 2.2. Front View-Interior of Bombardment Chamber
Bombardment Chamber Closes chamber with a solid piece of polycarbon-
Door (with Brace) ate plastic. Note the single, large o-ring which
seals vacuum in the chamber, and the self-positioning brace which eliminates flex of chamber
walls during bombardment cycle.
Rupture Disk Seals Rupture Disk against chamber end of Gas
Retaining Cap Acceleration Tube. This must be tightened securely.
The Torque Wrench is used in the holes in the cap
which are visible in the photo.
Microcarrier Launch Assembly Holds the DNA/microcarrier preparation on a
Macrocarrier sheet over the Stopping Screen in
the path of the helium shock wave.
Target Shelf Holds the biological target in a Petri plate in the
path of the accelerated DNA/microcarrier preparation. Particle flight distance is determined by positioning the shelf at one of four levels using slots in
the chamber walls.
11
Microcarrier
Launch Assembly
Power Switch
ON/OFF
Helium Pressure Gauge
Target Shelf
Bombardment
Chamber Door
Vacuum Gauge
Vac/Vent/Hold Switch
Vacuum/Vent Rate
Control Valves
Fire Switch
Disk Retaining
Cap
Table 2.3. Rear Connections
Refer to Figure 2.10 for a rear perspective view of the unit.
Helium Connection to Gas Tube Connects top of Gas Acceleration Tube to plastic
tubing from Solenoid Valve, supplying high pressure helium.
Over-Pressure Relief Valve Opens at 0.5 psi chamber pressure to relieve
accumulation of gas. A new safety feature.
Automatically resets after activation.
Vacuum Line, Chamber to Controls Supplies vacuum to chamber from control valves.
Helium Metering (Solenoid) Valve Supplies electric power to 3-way Solenoid Valve
Electrical Connection in helium line. Plug the three-pin connector from
the Solenoid Valve into this receptacle.
Line Cord Electrical Connection Supplies electric power to Biolistic unit from
house line. Plug connector from Line Cord into
this receptacle.
Vacuum Line, Connection to Source Connects unit to house vacuum supply or vacuum
pump via fittings included. Feeds into vacuum
flow controls.
Fig. 2.10. Rear view of main unit, component connection points.
12
Vacuum Port / Clamping Ring
Attachment Site
6.0 ft. PEEK Tubing
(connects to helium regulator)
2.5 ft PEEK Tubing
(connects to gas
acceleration tube)
Metering (solenoid)
Valve Power Cord
(connects to rear
of main unit)
Helium Pressure
Regulator
Adjustable
Metering Valve
Main Unit Power
Cord Socket
Over-Pressure Relief Valve
Vacuum (reinforced PVC)
Tygon®Tubing
Section 3
Installation
3.1 Connecting the PDS-1000/He System to a Helium Source
Refer to Section 2.2, Identification of Unit Controls and Components, prior to system
installation.
Helium Pressure Regulator Installation
Connecting the helium pressure regulator to a tank of pressurized helium.
Components needed:
• Pressure regulator for helium cylinder (with 0.45 micron in-line filter), provided with
unit (Figure 3.1).
• Cylinder of grade 4.5 to 5.0 helium (minimum 99.995% pure); maximum pressure of
2,600 psi, user supplied.
• 1 1/8" open-end wrench or a 10" or 12" adjustable wrench, user supplied.
Note: The regulator is intended for use only with helium gas under a maximum of 2,600 psi
of pressure. The outlet on pressurized helium cylinders used in the United States (maximum 2,600 psi) is compatible with the fitting supplied on the pressure regulator with the
PDS-1000/He unit (CGA 580, female fitting). Outside of the US, contact your local Bio-Rad
office for information regarding the proper cylinder/regulator fitting in your area.
Fig. 3.1. Helium regulator/tank attachment.
13
Helium Tank
Adjustment Handle
Attachment Site for
6 ft PEEK Tubing
Helium Regulator
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