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

5.8Distance 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 preor 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-cellu- lar 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).

Helium Regulator

Tank of Pressurized Helium

Microcarrier Launch

Assembly

(user provided)

Rupture Disk

Retaining Cap

Helium

Pressure

Regulator

Target Plate Shelf

6 ft PEEK Tubing

3-Way Helium Metering Vacuum (reinforced PVC) (solenoid) valve Tygon Tubing

2.5 ft PEEK Tubing

Fig. 1.1. Unit components, front view.

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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).

Before After

Gas Acceleration Tube

Rupture Disk

Macrocarrier

DNA-coated Microcarriers

Stopping Screen

Target Cells

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.

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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.

	Made in U.S.A.
Model No.	Serial No.
Voltage

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.

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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 includesSaccharomyces cerevisiae strain

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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.

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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)

2.5 ft PEEK Tubing To Unit

Power Cord

To Unit

6 ft PEEK Tubing To Helium Tank

Fig. 2.3. 3-way helium metering (solenoid) valve.

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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.

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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

Vacuum Tubing
	Rupture Disk
	Retaining Cap

Microcarrier

Launch Assembly

Fig. 2.5. PDS-1000/He main unit.

Fig. 2.6. Rupture disk retaining cap.

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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

A B

Fig. 2.7. Microcarrier launch assembly (A) and disassembled components (B).

Fig. 2.8. Target plate shelf.

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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 ViewExterior
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 atmo-
	spheric 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.

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Fire Switch

Vac/Vent/Hold Switch

Power Switch

ON/OFF

Vacuum Gauge

Vacuum/Vent Rate

Control Valves

Fig. 2.9. Front view of PDS-1000/He unit.

Helium Pressure Gauge

Bombardment

Chamber Door

Disk Retaining

Cap

Microcarrier

Launch Assembly

Target Shelf

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-posi-
	tioning 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 prepara-
	tion. Particle flight distance is determined by posi-
	tioning the shelf at one of four levels using slots in
	the chamber walls.

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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 pres-
	sure 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

Helium Metering (Solenoid) Valve Electrical Connection

Supplies vacuum to chamber from control valves.

Supplies electric power to 3-way Solenoid Valve 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.

2.5 ft PEEK Tubing (connects to gas acceleration tube)

Helium Pressure

Regulator

Adjustable

Metering Valve

Metering (solenoid) Valve Power Cord (connects to rear of main unit)

6.0 ft. PEEK Tubing (connects to helium regulator)

Over-Pressure Relief Valve

Main Unit Power

Cord Socket

Vacuum Port / Clamping Ring

Attachment Site

Vacuum (reinforced PVC)

Tygon® Tubing

Fig. 2.10. Rear view of main unit, component connection points.

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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.

Helium Tank

Helium Regulator

Adjustment Handle

Attachment Site for 6 ft PEEK Tubing

Fig. 3.1. Helium regulator/tank attachment.

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