Perfusion Fast-Step User`s Manual
Contents
1. Jie Zheng and Fred Sigworth Selecting Changes during Activation of Mutant Shaker Potassium Channels J General Physiology vol 10 August 1997 101 117 Rockefeller Univ Press Publication 5707 001 REV A www warneronline com 2 Perfusion Fast Step Model SF 77B Warner Instruments Sample Experiment As an example of the use of this stimulating system we may take an exper iment in which the activity of several adrenergic agonists and antagonists are investigated for their ability to modulate a K current in Pancreatic islet 8 cells Prior to beginning the experiment the reservoirs feeding the three pipes we will call them 0 1 and 2 are mounted approximately 1 meter above the microscope stage They do not have to be placed inside the Faraday cage For each pipe one of the six lines will contain physiological buffer i e the control bathing solution Pipe 0 will also have an adrenergic ago nist clonidine at 5 concentrations from 1 nM to 1 uM Pipe 2 will have the antagonist yohimbine also at 5 concentrations Pipe 1 will have epinephrine at 5 concentrations Thus each pipe has buffer and either an agonist or antagonist at 5 different concentrations After each of the solutions is brought into the manifold and through the pipes the buffer is turned on in each pipe to completely wash away the other solutions Thus at the beginning of the experiment each of three pipes has only buffer in it All solutions are turned off so that noth2. Publication 5707 001 REV A www warneronline com 5 Perfusion Fast Step Model SF 77B Warner Instruments Set Up Procedure Conto solution feeder lines the SF 77B Perfusion Fast Step can be reliably used with less than 1 ml of solution in the reservoir and with less than 0 5 ml to prime the line depending on distance of solution reservoir from the micro scope stage Loading Solutions It is recommended that one of the solutions in each of the three pipes should be the control buffer bathing the cells or patches This solution can be used as a rinsing solution between applications Solutions are poured into the syringes with the luer stopcock set to the closed position A useful method for the removal of air bubbles is as follows Remove the tip of a cotton tipped applicator and pack into the needle this acts as a bubble trap Even with careful removal of air bubbles prior to the solution reaching the PE tubing occasionally small bubbles will form The cotton tip breaks up the bubble then the smaller ones formed can be easily tapped out To prevent bubble formation in the bulk of the solution always pre warm solutions This entire procedure is made much easier by removing the manifold from the holding bar and unscrewing the tail portion of the manifold that is the section of the manifold through which solutions flow out and into the cap illary Because the output tubing is so much smaller than the input tubes the resistance
3. 25 1 4 mm OD 5 cm long 64 0811 Theta Glass Tubes 2 0 mm OD 10 cm long 100 64 0124 Glass Holder for 3SG700 5 or SG800 5 Glass 1 64 0125 Glass Holder for 2 mm Theta Glass 1 64 0126 Glass Holder for SG1000 5 Glass 1 Other sizes of square glass tubing are available from Vitrocom Inc Mountain Lakes New Jersey 07046 Telephone 201 402 1443 Publication 5707 001 REV A www warneronline com Perfusion Fast Step Model SF 77B Warner Instruments Warranty amp Services Please direct your questions regarding service or repairs to our engineering department at 800 599 4203 Warner Instrument Corp warrants the model SF 77B Perfusion Fast Step system to be free from defects in materials and workmanship for a period of 2 years from date of shipment If a failure occurs within this period we will either repair or replace at our option the faulty component s Publication 5707 001 REV A www warneronline com
4. 0 to pipe 1 for a period of time and then to pipe 2 returning either to pipe 1 or pipe 0 The stay in each pipe is independ ently variable It is also important to note that in the case of electrophysi ological experiments membrane potential may be varied independently of the pipe movement Thus this simple arrangement allows the experimenter the flexibility to build quite complex stimulus parameters Publication 5707 001 REV A www warneronline com 7 Perfusion Fast Step Model SF 77B Warner Instruments Specifications Number of Steps 1 to 7 2 to 8 positions Step Size Adjustable from 100 pm to 1 5 mm in 100 um increments with STEPS POSITION switch Step Speed Typically 20 msec for a 700 um step Step Control Manual 8 position with POSITION selector Analog Signal 5 positions with voltage levels 0 4 Volts 1V step Digital Signal 8 positions with 3 byte TTL signal Maximum Stepped 12 5 mm Range Mounting Handle 6 3 mm dia X 10 cm long Stepper Weight 110 5 grams including handle Solution Manifolds Three manifolds are supplied with each system MM series for SF 77B and SF 77BST and ML series with SF 77BLT Manifolds have 2 4 or 6 inputs Specify your choice when ordering MM Series MM 2 MM 4 and MM 6 manifolds use PE 50 tubing at the input and PE 10 tubing at output ML Series ML 2 ML 4 and ML 6 manifolds use PE 50 tubing at both input and output Solution Flow Rates With MM Series 100 pl min With ML
5. SF 77BST 1 msec Stepping with Theta Tubing The SF 77B has been successfully tested using theta style capillary tubing with a step speed of 1 msec between adjacent barrels Standard 2 mm diameter theta tubing is pulled to a tip diameter of 300 mm with a spacing of 100 mm between barrels In this design placement of the pipette tip is critical due to the smaller perfusion stream Since mechanical artifacts can be evident when using these small step sizes the motor drive voltage is adjustable at the rear panel Easy Set Up The stepper mechanism is compact lightweight and free of either mechanical or electrical noise The mechanism connects to the control box with a 2 meter shielded cable and is provided with a mounting rod for attachment to a manipulator Manifolds can support 2 4 or 6 inputs depending on the experiment Solutions flow from reservoirs to the manifold through PE 50 solutions arse Solutions B1 B6 Solutions C1 C6 tubing and PESO ings jka A A ding new reservoirs PE 10 tubing is i used to con Manifold A Manifold B Manifold C nect the mani fold outputs to Approximately 5 seconds for new solution flow when the glass tubes switching manifold input PE 10 Tubings Glass Tubes SP Stimulus effect in 20 milliseconds for 800 um step size Cell attached electrode A A Step XN Publication 5707 001 REV A www warneronline com 2 Perfusion
6. apply Do Not Dispose Product with Municipal Waste e Special Collection Disposal Required 2 Perfusion Fast Step Model SF 77B Warner Instruments Table of Contents Ta tea UT0 1707 MMM 2 3 Front Panel Description sss sisintsicsasissstesnntvaesettadonstitecuervanestdnumnnd 4 Rear Panel Description rurararavavaenennananannnnnnaanaanananase 5 System Description aniicitiiscieniimioieninnitinenindcinand 6 Set up Procedure ssssssssssssnesressnnsnunnunennnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnan 7 Capillary Tubes and Holder n 7 8 Mantol S oai duane incutiaitnctaunctmiamnnieniars 8 Mounting the Motor Assembly 9 10 Loading SOMMONG scdcscesccesntstvarsecrvstensivsntetieniecss 10 11 Care And Maintenance cccccssesseeeseeeees 11 Using the Controller mrsenranna 12 13 Fast Stepping with Theta Glass 13 Sample Experiment sacessescicsneatecscssercsassasieostssscssteestorensesatts 14 15 SpecificationS 4 l mmastik kaka 16 17 Accessory Reorders riva ntsessivistusavsittt vikaarist 18 Warranty amp Services istutan amvmatkusvisttsjninrkan veikals nmin 19 www warneronline com Perfusion Fast Step Model SF 77B Warner Instruments Introduction The SF 77B is a simple but highly effective stimulus solution delivery device for use in a variety of patch clamp and electrophysiology studies Control and test solutions flow co
7. is considerable and this can cause solution to back up into neighboring tubes during the filling procedure Without the output section of the manifold this does not occur After all the lines are filled the manifold can be screwed together again and mounted back in the holder Next remove the needle from the syringe Hold the needle about 1 2 mm below the syringe then open the luer valve Solution will flow under the force of gravity without the requirement for additional force for example by plunging the syringe As soon as the solution reaches the end allow a small drop of solution to enter the needle When the cotton is wet quickly insert the needle on the syringe Observe the solution passing along the PE 50 tubing then after making sure that there are no columns of air in the tubing switch the luer valve to closed Inspect the needle to see if any air bubbles are present tap the needle to dislodge Often large bub bles will form at the junction of the syringe and the luer valve these can be easily removed by tapping If this will not suffice then insert a long needle into the solution and draw out the air bubble care must be taken not to cross contaminate If at this point even a small bubble remains tap the syringe and dislodge This can be rather time consuming depending on the number of solutions to be used but is a very useful exercise that will max imise the performance of the SF 77 Perfusion Fast Step Publication 5707 0
8. 01 REV A www warneronline com 5 Perfusion Fast Step Model SF 77B Warner Instruments Set Up Procedure Conto Next attach the PE 50 tubing to the mainfold Open the luer valve and allow solution to flow through the glass pipes If at this point there is inhi bition of solution flow into the glass pipe turn the luer valve to closed and remove the outflowing section of the mainfold Insert into a piece of tygon tubing A D 1 16 O D 1 8 Wall 1 32 attached to a syringe and simply aspi rate and eject air Re attach the manifold and open the luer valve If there is no solution flow at this point apply positive pressure to the reservoir syringe making sure all other feeder lines are closed otherwise back flow will occur The luer valve should be briefly re opened to be sure that solution is indeed flowing through the line The same procedure is repeated for each solution in turn After all six solutions have been readied in this way it is advisable to turn on the buffer solution line so as to clean the capillary pipe of any residual solutions The same procedure should be repeated for each of the three capillary pipes When completed all the solutions to be used in the experiment are at the manifold so that simply turning a stopcock to the on position will allow that solution to flow from the manifold to the capillary tube This should require less than 2 seconds but 5 seconds are recommended to be sure that the last solutions
9. Fast Step Model SF 77B Warner Instruments Front Panel Description INSTRUMENT CORPORATION A STEPS POSITION Step length distance between stepper positions is selected with the thumbwheel control Minimum step size is 100 um 004 inches with the thumbwheel set at 1 and the maximum step is 1 5 mm 060 inches with the control at 15 When using the 3 barrel glass supplied with the standard SF 77B p n 3GC700 5 barrel to barrel spacing of 700 um the control is set to 7 7 x 100 um B MODE SELECT SWITCH Control of the stepper device can be INTERNAL manual EXT DIGITAL or EXT ANALOG The 3 position toggle switch is used to select the desired mode C INTERNAL A 5 position switch is used to manually select up to 5 positions An LED dis play indicates the position selected This same display indicates position when control is from an external signal EXT DIGITAL See rear panel description for controlling with a digital signal D EXT ANALOG An analog signal for stepper control is applied to this input Up to 5 posi tions can be used Positions correspond to the dc voltage levels as shown Position 0 0 volts Position 3 3 volts Position 1 1 volt Position 4 4 volts Position 2 2 volts E INITIALIZE The INITIALIZE push button is used to initialize the pipe position at the beginning of an experiment or at any time one wishes to check or confirm positions This is done in the INTERNAL manual mode by selecting the desir
10. Perfusion Fast Step Model SF 77B WARNER INSTRUMENTS A Harvard Apparatus Company Publication 5707 001 REV A WEEE RoHS Compliance Statement EU Directives WEEE and RoHS To Our Valued Customers We are committed to being a good corporate citizen As part of that commitment we strive to maintain an environmentally conscious manufacturing operation The European Union EU has enacted two Directives the first on product recycling Waste Electrical and Electronic Equipment WEEE and the second limiting the use of certain substances Restriction on the use of Hazardous Substances RoHS Over time these Directives will be implemented in the national laws of each EU Member State Once the final national regulations have been put into place recycling will be offered for our products which are within the scope of the WEEE Directive Products falling under the scope of the WEEE Directive available for sale after August 13 2005 will be identified with a wheelie bin symbol Two Categories of products covered by the WEEE Directive are currently exempt from the RoHS Directive Category 8 medical devices with the exception of implanted or infected products and Category 9 monitoring and control instruments Most of our products fall into either Category 8 or 9 and are currently exempt from the RoHS Directive We will continue to monitor the application of the RoHS Directive to its products and will comply with any changes as they
11. Series 1 ml min Rates are measured with a solution reservoir height of approximately 60 cm 24 inches Control Box Size 6 3 cm H x 14 cm W x 23 cm D Power Requirements 100 130 or 220 250 VAC 50 60 Hz 10 VA System Shipping 2 7 kg Weight Warranty 2 years parts and labor Publication 5707 001 REV A www warneronline com Perfusion Fast Step Model SF 77B Warner Instruments Specifications contd Glass Tubes end views 3SG700 5 G800 5 SG1000 5 TGC200 10 boos 0 mi pr wp 0 ALL DIMENSIONS IN MILLIMETERS Publication 5707 001 REV A www warneronline com 4 Perfusion Fast Step Model SF 77B Warner Instruments Accessory Reorders Business hours are 8 30 AM to 5 00 PM eastern time Monday to Friday Telephone 800 599 4203 Fax 203 776 1278 The following accessories are available from stock Order No Description Pkg Qty 64 0200 Manifold 2 to 1 1 64 0201 Manifold 4 to 1 1 64 0202 Manifold 6 to 1 1 64 0203 Manifold 2 to 1 1 64 0204 Manifold 4 to 1 1 64 0205 Manifold 6 to 1 1 64 0750 Polyethylene Tubing 10 64 0752 Polyethylene Tubing 10 64 0119 3 Barrel Square Glass Tubes 0 6 mm ID 10 5 cm long 64 0120 3 Barrel Square Glass Tubes 0 6 mm ID 10 10 cm long 64 0121 Single Barrel Square Glass Tubes 0 6 mm ID 25 0 84 mm OD 5 cm long 64 0122 Single Barrel Square Glass Tubes 1 mm ID
12. adjust the flexture spring holder to correct the alignment if neces sary loosen the collar set screw and move the collar to align the flex ture spring to its neutral unflexed position External Control If the initialization is performed when there is 0 volts on the input line then the pipes will automatically assume the 0 position If there is another voltage on the line then the pipes will go to that position immediately and automatically after the initialization If at any time during an experiment or series of experiments the posi tions become confused then simply pushing the initialize button will bring everything back into register Fast Stepping with Theta Glass Very fast perfusion stepping is possible using 2 barrel Theta tubing The technique requires close attention to detail and careful placing of the pipes and the excised patch The following is information provided as a general guide Preparation of the Theta Glass The tubing is pulled on a standard puller for a tip diameter of approx imately 300 um and a barrel to barrel spacing of approximately 100 um Motor Voltage Adjustment When using 100 um steps it is important to minimize any vibration produced by the stepper motor This is accomplished by reducing the motor voltage with the control located on the rear panel The voltage is lowered until the vibration artifact is minimized Any residual artifact may be removed by subtracting averaged null traces Reference
13. ance traveled is adjusted with the counter control steps position Minimum step is 0 1mm 1 step and maximum is 1 5mm 15 steps For the standard 3SG700 5 capillary pipes supplied barrel to barel spacing 0 7 mm a step length of 7 is required to move from center to center This adjustment should however be made empirically for each new set of capillary pipes Duration When driven with an analog signal the time spent in a posi tion is determined simply by how long the voltage corresponding to that position lasts To move from pipe 0 to pipe 1 for 1 second and then return to pipe 0 requires a voltage step of from 0 to 1 V that lasts 1 second and then returns to OV I If a digital signal is the external control the stepper will stay in a posi tion until it receives the next command In the absence of a signal the motor will go to the O position Manually the position is maintained until changed to another switch position Publication 5707 001 REV A www warneronline com 4 Perfusion Fast Step Model SF 77B Warner Instruments Set Up Procedure Conto Initializing the Position Before beginning any experiment it is required to initialize the pipe position by pressing the Initialize button The stepper motor will move to whichever position is manually selected This brings the pipes into a known position from which all other positions are refer enced For a setup with 3 pipes initialize in the center position 2 Re
14. ce Make sure the rubber seal is inside the head before screwing the parts together ML Series Manifolds These manifolds use the PE 50 tubing on both input and output The larger PE 50 fits nicely into the G1000 5 glass pipes used with the SF 77BLT Publication 5707 001 REV A www warneronline com s Perfusion Fast Step Model SF 77B Warner Instruments Set Up Procedure Conto Mounting the Motor Assembly The SF 77B Perfusion Fast Step is designed to be mounted on and posi tioned by a manipulator It is light enough to be attached to a microma nipulator without producing drift but for most applications a standard coarse manipulator will be sufficient Ideally the device should be posi tioned within the set up so that it can be easily removed from the manipu lator and replaced This will facilitate cleaning and loading the device Positioning the angle of the motor assembly is made by loosening the black wing nut When loosened the motor mount bracket and manifold bar can be rotated to accomodate the setup Additionally the angle of the motor extension bar white plastic bar attached to the motor drive can be adjust ed by rotating the motor collar black block mounted on the motor Before rotating the block loosen the small set screw with the Allen wrench sup plied with the SF 77B motor assembly Retighten the screw lightly once the desired position is attained NOTE Do not overtighten this screw Motor operation may b
15. cted from your data acquisition program Manually the system can be stepped to 8 positions in 7 equally spaced steps These same 8 positions can also be directly selected by applying an analog signal to the external analog input BNC or by passing a 3 byte word to the TTL input on the instrument rear panel Square Glass Stimulus Ports The square glass tubes used for solution delivery significantly reduces mixing turbulence between solutions allowing the SF 77B to be used for studies with both membrane patches and whole cells even when the cells are not fixed to a substrate Publication 5707 001 REV A www warneronline com s Perfusion Fast Step Model SF 77B Warner Instruments Introduction Contd System Versatility The design of the SF 77B permits the use of various size glass tubing for perfusion delivery SF 77B Standard System 0 7 mm ID tubes The standard system is shipped with 3SG700 5 single walled 3 barrel glass tubing which eliminates the need to glue individual barrels together Spacing between barrels is 0 7 mm and step speed between adjacent barrels is typically 20 msec Single barrel SG800 5 tubes up to 5 can be used with the same holder SF 77BLT Large Tube System 1 0 mm ID tubes Larger stimulus ports are required when using the SF 77B with larger cell structures such as the Xenopus oocyte Solutions are delivered through 1 0 mm ID square tubes SG1000 5 with barrel to barrel spac ing of 1 4 mm
16. e adversely effected A subsection of this part of the device is the assembly of tubing etc that will feed solutions into the manifolds and then to the pipes Most simply the feeder arrangement consists of up to 18 syringes with luer stop cocks ideally 2 way to minimize three way can be used however this tends to introduce air bubbles at the point of entry into the PE 50 tubing Be careful to avoid air bubbles as once in the PE tubing the flow of solution is inhibited Depending on the rate of flow used for a particular application this may not be detected immediately Avoidance and removal of bubbles will be discussed later The syringes are mounted in a rack loading end up and placed approximately 1 meter above the surface on which the chamber is fastened usually the microscope stage The height above the device will depend on the experimental design for exam ple perfusing an inside out patch will require a slower flow rate than per fusing a cell The syringes can be mounted either inside or outside of the Faraday cage if one is being used A distinct advantage to this system is that electrical noise is not a problem even for single channel recording Syringes of 20 or 60 ml volume are recommended although other containers such as seperatory funnels or bottles can be used The main advantage of syringes is their economy and disposability For expen sive reagents smaller syringes can be used With careful preparation of the
17. e Solution changes can be made in as little as 20 50 ms approxi mately 1 ms using 2 barrel Theta tubing and once changed the new solu tion with its particular concentration of pharmacological agent will remain stable until changed again An important capability of this device is also that a large number of solu tions can be utilized during the course of a single experiment in spite of the compact size of the device This takes a bit of organizational thought before starting the experiment but the return is the ability to easily use 15 30 different solutions in a single experiment This is accomplished by having as many as six solutions feeding through a unique manifold into each of the three pipes in front of which the cell may be placed This allows any of the three pipes to have any one of 6 solutions flowing through it at any time It is even possible during the course of an experiment to change the six solutions flowing into any one of the manifolds allowing a virtually limitless number of solutions to be used In order to fully implement this feature it is important to realize that there are actually three time frames that need to be considered in the proper use of the device The first is the 50 ms or less time course for changing between different pipes The second is the 5 second dead time required to change from one solution to another in the same pipe Finally there is the 30 60 second time required to change one of the six s
18. ed position and depressing the push button Publication 5707 001 REV A www warneronline com s Perfusion Fast Step Model SF 77B Warner Instruments Rear Panel Description SF 77B FUSE AT 0 25 0 125 AMPS Perfusion FOR 100 125 220 240 VAC Fast Step 5X20mm FUSE VOLTAGE ADJUST WARNER INSTRUMENT CORPORATION FE F POWER BLOCK This block includes the power line cord connector ON OFF switch and the input power fuse G MOTOR The stepper mechanism connects to the control box here H EXTERNAL DIGITAL INPUT Digital inputs for position control connect to the 3 BNC connectors labeled DO D1 and D2 Up to 8 positions can be controlled when all 3 lines are used Digital inputs are TTL compatible and readily accept digital outputs from computers and computer interfaces Positions are controlled per the table shown here TRUTH TABLE Position DO D1 D2 0 L L L 1 H L L 2 L H L 3 H H L 4 L L H 5 H L H 6 L H H 7 H H H J MOTOR VOLTAGE ADJUST This control is factory set and only used for experiments involving small 100 um steps Its use is further described in the section dealing with Fast Stepping with Theta Tubing Publication 5707 001 REV A www warneronline com 4 Perfusion Fast Step Model SF 77B Warner Instruments System Description The main use of the SF 77B Perfusion Fast Step is the rapid change of solutions bathing a single cell or patch of membrane attached to a patch electrod
19. have been completely washed out Although in general there is no leakage from the end of the pipes we recommend that at the start of an experiment all three pipes have buffer flowing through them see sample Experiment below If a line is not to be used e if less than six solutions are to be used in any one pipe then it is necessary to use a plug for that line on the manifold This is to prevent solution from backing up into an open line Alternatively all unused lines can simply be loaded with buffer solution however this is more time consuming NOTE It is best to keep the output ends of the pipes submerged in solution or water when changing the setup If the pipes are left unsubmerged for any length of time fluid will weep from the tubings and air bubbles will creep in Care and Maintenance At the end of each experiment allow all solutions to flow out of the reser voir and add a few ml of 70 ethanol The manifold is composed of delrin and stainless steel and PE tubing is unaffected by ethanol thus this cleaning procedure can be withstood Next flush all the lines with water After all solutions have run through remove the needle and dispose of the cotton tip prevent cross contamination of solutions Finally air dry pressurized solution lines Publication 5707 001 REV A www warneronline com 3 Perfusion Fast Step Model SF 77B Warner Instruments Set Up Procedure Conto Using the Controller Control of
20. ing is leaking from the pipes Finally the position of the pipes is initialized After a cell is chosen and the whole cell patch clamp configuration obtained the stimulating apparatus is lowered into the dish and under low power is manipulated into the region of the cell Depending on the prepa ration the cell may either remain attached to the dish substrate or may be suspended above the floor of the dish on the tip of the electrode In either case the pipes are manipulated into a position in which the cell is directly opposite the lumen of pipe 0 Buffer is turned on in all three pipes so that the cell is now in a flowing solution of control physiological buffer A series of 5 membrane voltage pulses each lasting 50 ms are delivered to deter mine the magnitude of the K current under control conditions Following this the solution in pipe 1 is switched to the lowest concentra tion of epinephrine A stimulating protocol in which the cell is moved to pipe 1 by delivering a 1 volt step from the analog output immediately after which a series of membrane voltage steps lasting 50 ms each are deliv ered and then the cell is returned to pipe 0 by changing to 0 volts from the output The duration of the step is 300 ms 5 x 50 ms with a 10 ms delay between each membrane voltage step The data are saved and the solution in pipe 0 is changed to the next highest concentration of epi nephrine by turning one luer stopcock to the off position and the next
21. nsions of your partic ular set up and chamber Ideally however it is best to have the tubes more or less centered on the holder The tubes and holder can be attached easily to the mounting device but this should be done after this device is installed as described in the next section Manifolds Manifolds are made with 2 4 and 6 inputs specified at time of order MM Series manifolds are standard with the SF 77B and SF 77BST ML Series are provided with the SF 77BLT MM Series Manifolds The inputs connect to PE 50 tubing which fits over the hypodermic tubings A length of PE 10 tubing is factory installed in the manifold output This tubing fits into the glass pipes and is sealed with wax At some time in the future the PE 10 tubing will require replacement Proceed as follows 1 Unscrew the manifold tail from the head and pull the old tubing from the tail 2 Cut a length of PE 10 tubing 8 to 9 inches long 3 You need to stretch one end of the tubing so that its diameter is reduced sufficiently to fit into the hole in the tail piece Do this by gripping one end between thumb and forefinger of both hands and pulling firmly This may take a little practice 4 Trim off the unstretched portion of tubing at the end so that the smaller diameter portion can be inserted into the hole Pull the tubing through until the full diameter is inside the tail Trim the tubing flush with the tail inside end and reinstall in the head pie
22. ntinuously through adjacent stimulus delivery tubes and a stepper mechanism selects which tube is directed at the prepa ration The rapid response and nominal hysteresis of the stepper mecha nism allows for very fast switching times Complete solution changes are typically achieved within 20 msec for a large 700 mm step and times are significantly shortened as the step size is decreased Multiple Solution Studies In the standard configuration up to six different solutions are con nected to a single input manifold which in turn is connected to one of three square glass stimulus ports The three stimulus port design is superior to a two port design in that complex solution exchange pro tocols can be brought to bear on the sample under study Since the complete system is designed o accommodate three manifolds one for each stimulus port and each manifold can accommodate up to 6 feed lines it is possible to immediately select between 18 different input solutions e Solution changes between stimulus ports occur within milliseconds e Changes between solutions connected to an individual port occur within five seconds e Entirely new solutions can be added into any port with a wait ing time of no more than 30 seconds e The cell is never required to pass through intervening solutions to get from control to test solution Manual or External Control The stepper mechanism can be manually controlled via the front panel or externally dire
23. one to the on position 5 seconds later the same protocol can be repeated now examining the effect of the higher concentration of epinephrine on the K current elicited by voltage After the series of 5 concentrations of epinephrine are completed the pipes are shifted so that the cell is now in the lumen of Pipe 1 which has Publication 5707 001 REV A www warneronline com 4 Perfusion Fast Step Model SF 77B Warner Instruments Sample Experiment Conto been changed to buffer This is accomplished by having 1 volt appear on the output The same experiment can now be repeated with different con centrations of clonidine as the pipes are moved so that the cell goes from pipe 1 to pipe 0 The only change in the stimulus protocol is that the volt age output goes from 1 to 0 and back to 1 for each test series Upon completion of the agonist series the antagonist effects can be tested First the lowest concentration of Yohimbine is turned on in pipe 2 and the cell is moved to that pipe i e the pipes are shifted so that the cell is in front of this pipe An effective concentration of epinephrine determined from the earlier run is now turned on in pipe 1 For this experiment the cell is shifted from pipe 2 to pipe 1 but possibly for only a single 50 ms voltage pulse so that the antagonist cannot be competed off by the agonist Thus the protocol is for the analog output to go from 2 volts to 1 volt and back to 2 volts in about 90 ms Once in
24. s supplied with each system The stan dard SF 77B holder is the GH 1 It holds the 3SG700 5 three barrel tub ing or up to 5 individual barrels of SG800 5 tubing 0 6 mm inside 0 12 mm wall SF 77BLT is supplied with the GH 10 holder which holds up to 3 of the SG1000 5 glass tubes 1 0 mm inside 0 2 mm wall Holder GH 2T holds the 2 mm Theta glass Holding the Glass The capillaries can be mounted directly in the holder which maintains them in position by pressure The 3 barrel glass is simply placed into the holder slot and the threaded handle is tightened to hold the glass rigid If single tubes are used we recommend that the capillaries be cemented together first to insure that they are perfectly level and in alignment Place the capillaries on a flat surface and butt the ends against a straight edge Alignment is critical We suggest you check under a dissecting microscope Use only a small dab of cement placed near but not exactly at the center line in the lengthwise direction Do not put the cement near either end of the tubes as capillary action will carry it into the tubes and may clog them Publication 5707 001 REV A www warneronline com 3 Perfusion Fast Step Model SF 77B Warner Instruments Set Up Procedure Conto After the cement dries a few minutes is sufficient simply slide the tubes into the holder such that nearly half of the tube length extends from the holder This length is adjustable depending on the dime
25. tarting solutions flow ing into any of the three manifolds The third time frame is only important for experiments that would typically use more than 15 different solutions in a single experiment Each of these uses will be explained in detail and a typical experiment will be worked through using each of these features Publication 5707 001 REV A www warneronline com Perfusion Fast Step Model SF 77B Warner Instruments Set Up Procedure The SF 77B Perfusion Fast Step systems are packaged in three parts 1 stepper motor assembly with a set of 3 manifolds 2 the electronic controller 3 glass holder the capillary tubes and polyeyhylene tubing MM Series are supplied with SF 77B and SF 77BST ML Series are sup plied with SF 77BLT Manifolds are available in 2 4 and 6 inputs The Capillary Tubes and Holder Glass Capillaries The SF 77 B Perfusion Fast Step makes use of square capillary tubes for its pipes These facilitate placement of the tubes in the chamber and more importantly reduce turbulence and provide a more even flow of solution The capillaries supplied with the standard SF 77Bare 3 barrel which have an inside dimension of 0 6 mm each barrel and a wall of 0 1 mm The barrels have been fused together in manufacture so no gluing is necessary Larger 1 0 mm tubing is supplied with system SF 77BLT and 2 barrel Theta tubing is supplied with the SF 77BST Glass Holders The appropriate glass holder i
26. the SF 77B Perfusion Fast Step has been designed to be as simple as possible The device can be controlled manually or by elec tronic signals generated by a computer or stimulator The three main parameters under the investigators control are position step size and dura tion Position tells the device which capillary pipe to position in front of the target cell or membrane patch Step size adjustable from 0 1mm to 1 5mm in 0 1mm steps is selected for the center to center distance of the pipes Duration determines the length of time that the capillary pipes remain in a given position Position There are five available positions in INTERNAL manual and EXT ANALOG modes and eight positions in the EXT DIGITAL mode The typical use is with 3 pipes and only three positions are required Position is select ed by manual switch position or by an external analog or digital signal The position selected is displayed on the LED readout Analog Voltage Control When using an analog input the positions are voltage sensitive Zero volts corresponds to 0 position and stepping the voltage in 1 volt increments steps the device 2 volts position 2 etc Digital Control The rear panel has 3 BNC inputs to accept standard TTL compatible signals for digital control from a computer or other digital signal source The 3 inputs allow for as many as 8 positions to be controlled See REAR PANEL DESCRIPTION section 2 0 for more detail Step Size The dist
27. the agonist solution change time is about 30 ms the voltage pulse is delivered to evoke the K current and the cell is immediately moved back to pipe 2 This can be repeated for the 5 concentrations of antagonist available to pipe 2 For this experiment all of the protocols could have been written previous ly requiring only a new protocol to be brought up one or two luer stop cocks to be turned on or off and a button to be pushed activating the com puter acquisition The total estimated time of such an experiment could be as little as 4 5 minutes In the course of this relatively straightforward experiment 15 concentra tions of 3 pharmacological agents have been tested for their effects on the K current These data should provide relatively complete dose response data for these three agents and by the simple process of changing the dura tion of the steps into the stimulus solutions by simply lengthening the volt age output kinetic data could also be obtained If the effective concentra tions for these drugs had already been known a smaller range of concen trations could have been used and additional drugs included in the experi ment New reservoirs could also be installed with new drugs extending the time of the experiment only an additional 10 or so minutes More complex stimulus protocols are also possible in which there are sev eral shifts of the pipes during the course of a single test Thus it would be possible to go from pipe
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