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.
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.
Table of Contents Section 1 1.1 1.2 1.3 1.4 Section 2 2.1 2.2 Section 3 3.1 3.2 3.3 Section 4 4.1 4.2 4.3 Section 5 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 Section 6 6.1 6.2 6.3 6.4 6.5 6.6 Section 7 Biolistic PDS-1000/He Particle Delivery System..............................................1 Particle Delivery Technology ....................................................................................1 Overview of PDS-1000/He Particle Delivery System .............................................
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.
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.
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).
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.
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.
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.
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 soleno
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.
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 B A Fig. 2.7. Microcarrier launch assembly (A) and disassembled components (B). Fig. 2.8.
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.
Helium Pressure Gauge Fire Switch Vac/Vent/Hold Switch Power Switch ON/OFF Bombardment Chamber Door Vacuum Gauge Disk Retaining Cap Microcarrier Launch Assembly Target Shelf Vacuum/Vent Rate Control Valves Fig. 2.9. Front view of PDS-1000/He unit. Table 2.2. Front View-Interior of Bombardment Chamber Bombardment Chamber Door (with Brace) Closes chamber with a solid piece of polycarbonate plastic.
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.
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.
1. Secure the cylinder to a wall, post, or other anchored fixture so it will not tip or fall during use. 2. Inspect the cylinder valve for dirt, dust, oil, grease, or damaged threads. Remove dust and dirt with a clean cloth. Do not attach the regulator if you determine that the valve port is damaged or cannot be cleaned. Inform your gas supplier of this condition and request a replacement cylinder. 3.
3.2 Connecting the PDS-1000/He Unit to a Vacuum Source The main unit is shipped with the vacuum hose assembly attached to the port on the rear of the unit, via the clamping assembly. Only the connection of the free end of the tubing to a vacuum source is required for connecting the vacuum source to the system. Choice of Vacuum Source A vacuum system with a 3 cfm (cubic feet per minute) or 100 l/min pumping capacity is recommended for use with the PDS-1000/He unit.
Quick Flange Adaptor Centering Ring Vacuum tubing Male Pipe Adaptor Clamping Ring Fig. 3.2. Components used to connect vacuum source to main unit. 3.3 Power Cord / Voltage Regulator The power cord must be plugged directly into a 100 V-120 V outlet. Plug other end into unit. For use with a 220 V or 240 V line voltage (users outside the US or Canada), connect a voltage converter to the power cord prior to use (catalog number 165-2259, see Figure 3.3).
Section 4 Operation of the PDS-1000/He Instrument 4.1 Preparation of System Components Prior to Bombardment Instrument Preparation 1. Verify that helium tank has 200 psi in excess of desired rupture disk pressure for bombardment. 2. Set gap distance between rupture disk retaining cap and microcarrier launch assembly.
Three hexagonal gap adjustment tools of 1/8", 1/4", and 3/8" have been provided to reproducibly set the gap distance (Figure 2.4). A 1/4" distance between the rupture disk retaining cap and the macrocarrier cover lid is recommended for initial optimization bombardments. While holding the hexagonal gap adjustment tool against the bottom of the rupture disk retaining cap, turn the adjustable nest until the macrocarrier cover lid touches the gap adjustment tool.
3. Prepare the Rupture Disk Retaining Cap. After setting the gap between cap and microcarrier launch assembly, wrap the Rupture Disk Retaining Cap in aluminum foil and sterilize by autoclaving. 4. Prepare the Microcarrier Launch Assembly. The effect of the gas shock wave on the microcarrier velocities is determined in part by the gap between the rupture disk and the macrocarrier.
Consumable Preparation Several consumables are available for use with the PDS-1000/He system (Figure 4.3). B A C E A. Macrocarriers B. Rupture disks C. Stopping screens D. Tungsten microcarriers E. Gold microcarriers D Fig. 4.3. Consumables for the PDS-1000/He instrument. The 0.6 micron gold is not pictured. 1. Macrocarriers Pre-assemble and pre-sterilize the macrocarrier set in a macrocarrier holder prior to performing sample cell/tissue bombardments.
Direction of Tool Movement Macrocarrier Insertion Tool Helium Macrocarrier Helium Macrocarrier Holder Fig. 4.4A and 4.4B. Insertion of macrocarrier into macrocarrier holder with plastic insertion tool. 3. Stopping screens Transfer selected stopping screens (Figure 4.3) to individual Petri dishes for easier handling. Sterilization by autoclaving is recommended. Alternatively, these parts can be sterilized by soaking in 70% ethanol, followed by drying in a sterile environment. 4.
Coating Washed Microcarriers with DNA The following procedure is sufficient for six bombardments; if fewer bombardments are needed, adjust the quantities accordingly. Vortex the microcarriers prepared in 50% glycerol (30 mg/ml) for 5 minutes on a platform vortexer to resuspend and disrupt agglomerated particles. When removing aliquots of microcarriers, it is important to continuously vortex the tube containing the microcarriers to maximize uniform sampling.
Firing the Device 1. Plug in power cord from main unit to electrical outlet. 2. Power ON. 3. Sterilize chamber walls with 70% ethanol. 4. Load sterile rupture disk into sterile retaining cap. 5. Secure retaining cap to end of gas acceleration tube (inside, top of bombardment chamber) and tighten with torque wrench. 6. Load macrocarrier and stopping screen into microcarrier launch assembly. 7. Place microcarrier launch assembly and target cells in chamber and close door. 8.
4. Coating microcarriers with DNA The day of the scheduled bombardment, coat the microcarriers with DNA. To obtain the best results, use the DNA-coated microcarriers as soon as possible. 5. Loading DNA-coated microcarriers onto a macrocarrier/macrocarrier holder Each macrocarrier is placed inside a macrocarrier holder and sterilized, as described above.
7. Loading the rupture disk Unscrew the rupture disk retaining cap from the gas acceleration tube from within the bombardment chamber (Figure 4.6) or unwrap cap from sterile wrapping. Fig. 4.6. Removal/mounting of rupture disk retaining cap onto end of gas acceleration tube inside bombardment chamber. Select rupture disk of desired burst pressure. Handle all rupture disks with sterile forceps or Disk-Vac (catalog number 165-2323).
Screw the rupture disk retaining cap onto the gas acceleration tube using a left -to-right motion. Never tighten the rupture disk retaining cap without a rupture disk in place or scratching and deformation of the two metal surfaces will occur and cause helium to leak when a rupture disk is pressurized. The retaining cap is tightened to a torque of 60 inch/pounds with the retaining cap torque wrench. To use the torque wrench, insert the short end of the metal rod into an accessible hole in the retaining cap.
Fig. 4.9. Placement of stopping screen inside fixed nest with macrocarrier and cover lid removed. Fig. 4.10. Removal / replacement of macrocarrier cover lid with assembled fixed nest. Macrocarrier holder (with macrocarrier properly inserted) is inverted and placed atop fixed nest.
9. Target cells/tissue placement in chamber Place the Target Shelf at the desired level inside the bombardment chamber. Place the sample (usually contained within a Petri dish) on the Target Shelf. Close and latch the sample chamber door. 10. Chamber evacuation/hold Turn on the vacuum source. Set the vacuum switch on the PDS-1000/He (middle red control switch, Figure 4.11) to the VAC position. Evacuate the sample chamber to the desired level, at least 5 inches of mercury.
Estimate rupture disk burst pressure by observing the helium pressure gauge at the top of the acceleration tube. A small pop will be heard when the rupture disk bursts. The rupture disk should burst within 10% of the indicated rupture pressure and within 11–13 seconds. Release the FIRE switch immediately after the disk ruptures to avoid wasting helium. Releasing the FIRE switch prior to disk rupture will vent the gas acceleration tube via the 3-way helium metering (solenoid) valve.
Fig. 4.13. View of spent rupture disk within the retaining cap. 4.3 Removal of Residual Helium Pressure—Shut Down After completing all bombardment(s), remove the helium pressure from the PDS-1000/He system and close the helium cylinder valve. Perform the following steps to remove helium pressure from the system. 1. Close the helium cylinder valve and chamber door. 2.
Table 5.1. Cell Types, Settings, and Conditions Cell Type Growth Cell Phase Density Osmoticum Target Helium Vacuum Distance Pressure Particle (inches Hg) (cm) (psi) Size Bacteria Late log 108–109 per 0.75 M sorbitol to early 100 mm stationary plate 29 6 1,100 M5 tungsten Yeast Early 108–109 per 0.75 M sorbitol stationary 100 mm and 0.75 M plate manitol 28 6 1,300 0.6 µ gold Algae Log 108–109 per 100 mm plate 29 6 1,300 0.6 µ gold – 10 explants None per 100 mm plate 28 6 1,300 1.
Suggested starting helium pressure conditions for optimizing various biological systems are: Cell type Rupture disk pressure Bacteria Fungi Yeast Plant cells/tissue Mammalian cells 1,100 psi 1,300 psi 1,300 psi 1,100 psi 1,100 psi 5.4 Solenoid Valve Adjustment A factory pre-set metering valve (black knob) on the 3-way helium metering (solenoid) valve assembly controls the fill rate of the gas acceleration tube.
L1= 3 cm L2= 6 cm L3= 9 cm L4= 12 cm Fig. 5.1. Target shelf placement and corresponding target distances. 5.9 Microcarrier Selection Types of Microcarrier Two types of microcarriers are available, gold and tungsten. The density of these microcarriers is sufficient to penetrate a wide variety of cell and tissue types using the Biolistic PDS-1000/He acceleration system. Five tungsten particle sizes are available.
Recommended starting particle size/type for bombardment of various cell types is Bacteria Yeast Algae Plant cells/tissue Animal cell cultures Sub-cellular organelles 0.7 µm (M5) tungsten 0.6 µm gold 0.6 µm gold 1.0 µm gold 1.6 µm gold 0.6 µm gold Table 5.1 and bulletins 1688 and 2015 give a detailed discussion of optimization parameters. 5.10 Preparation of Biological Material for Bombardment Plant Cells Many factors contribute to optimum performance in any system for plant gene transfer.
Some kinds of embryogenic suspension cultures approach this ideal. In fact, it is this type of tissue which has allowed great success in the microcarrier-mediated transformation of recalcitrant species such as maize [see, for example, Gordon-Kamm et al., The Plant Cell, 2, 603-618 (1990)]. Most explants, however, differ from this ideal in substantive ways.
more appropriate to use an extractive assay procedure in which expression level can be measured, for example, as a function of enzyme activity per unit of total protein. Several excellent markers (and associated assay procedures) are available for this type of study. These include luciferase and ß-glucuronidase measured by a fluorometric process, and neomycin phosphotransferase, for which a commercial ELISA assay has recently been marketed.
Section 6 Troubleshooting 6.1 Rupture Disk Bursts at Incorrect Pressure Probable Cause 1. The helium flow rate is too fast. Action Note the helium burst pressure by observing the helium pressure gauge at the top of the main unit. A factory pre-set metering valve (black knob) on the external 3-way helium metering (solenoid) valve assembly controls the fill rate of the gas acceleration tube.
6.3 Excessive Gas Usage Probable Cause Action 1. Leaking helium fitting in the system Isolate the components from the He cylinder to the solenoid assembly by completely closing the metering valve (turning the knob counterclockwise until it stops.) Ensure that the regulator gauge maintains pressure over time. “Soap Test” the fittings by spraying a film of soapy water at the site of the fittings and looking for bubble formation. Tighten any fittings that appear to leak.
6.6 Unit Will Not Pressurize Gas Acceleration Tube Probable Cause 1. Rupture disk not in position or not sealed by retaining cap. Action Insert disk properly 2. Main cylinder valve of Helium tank not open Adjust according to Section 3.1 3. Insufficient vacuum Examine vacuum connections (see Section 3.2) 4.
Section 7 Product Information 7.
Catalog Number 165-2296 165-2297 165-2298 165-2322 165-2323 165-2326 165-2327 165-2328 165-2329 165-2330 165-2331 165-2332 165-2333 165-2334 165-2335 165-2336 170-3100 Product Description 500 Standard Pressure Kit, with 1.6 µ gold + 1,800 psi disks 500 Standard Pressure Kit, with 1.6 µ gold + 2,000 psi disks 500 Standard Pressure Kit, with 1.
Spare Part Number Product Description 910-9511 910-9515 910-9529 Helium Pressure Gauge Vacuum Gauge Wrench, Open End, 1/4" x 5/16" Metal Case Version (Discontinued) 800-3014 910-9549 910-9526 Complete Door Assembly Complete Filter Housing Filter Housing O-Ring Gunpowder Version (Discontinued) 910-9538 920-3009 910-0019 920-3061 910-9522 910-9530 910-9531 920-3022 920-3023 920-3026 920-3035 920-3058 920-3007 920-3008 920-3010 920-3011 920-3013 920-3014 920-3015 920-3016 920-3017 920-3018 920-3060 920-3
Section 8 Appendices 8.1 Cleaning the PDS-1000/He Device 1. Chamber Clean the chamber with 70% ethanol. Allow time for drying. 2. Gas Acceleration tube A. Unscrew the rupture disk retaining cap from the gas acceleration tube Do not autoclave or flame sterilize the PDS-1000/He. Autoclaving will wet electrical components, thus creating a potential electrical shock hazard. B. Disconnect the tubing assembly from the gas inlet on the rear of the acceleration tube (See Figure 1.2).
Aspects unique to the metal chamber design • Metal bombardment chamber. • Top slots of the metal case. These are used only for the target shelf placement when using gunpowder acceleration system. • Door construction: Gasket design changed. Portions removed that were dedicated to use with gunpowder acceleration. Door brace added in plastic case design for additional safety. • External vacuum filter assembly components. Located on rear and side of metal case unit.
Table 8.
8.3 Specifications General Specifications, PDS-1000/He Biolistic Particle Delivery System Dimensions 29 (width) x 25.5 (depth) x 47.5 (height) cm Construction Aluminum, ABS plastic and acrylic chassis Weight 15 kg Electrical input voltage 100-120 VAC, 50- 60 Hz Maximum current <5 Amps Mechanical Fuse 6.3 A, 250 V, 5 x 20 mm Vacuum <0.4 inches Mercury/minute leakage Over-Pressure 0.
8.4 Performing a Bombardment Quick Guide Before the Bombardment 1. Select/adjust bombardment parameters for Gap distance between rupture disk retaining cap and microcarrier launch assembly. Placement of stopping screen support in proper position inside fixed nest of microcarrier launch assembly 2. Check helium supply (200 psi in excess of desired rupture pressure). 3. Clean/sterilize: Equipment: rupture disk retaining cap, microcarrier launch assembly Consumables: macrocarriers/macrocarrier holders 4.
Bio-Rad Laboratories Molecular Bioscience Group 2000 Alfred Nobel Drive Hercules, California 94547 Telephone (510) 741-1000 Fax: (510) 741-5800 M1652249LEASE Rev D Australia, Bio-Rad Laboratories Pty Limited, Block Y Unit 1, Regents Park Industrial Estate, 391 Park Road, Regents Park, NSW 2143 • Phone 02-9914-2800 • Fax 02-9914-2888 Austria, Bio-Rad Laboratories Ges.m.b.H., Auhofstrasse 78D, 1130 Wien • Phone (1) 877 89 01 • Fax (1) 876 56 29 Belgium, Bio-Rad Laboratories S.A./N.V.
