STREX Cell Strain Instrument User Manual
Contents
1. Digit Degree of stretch Distance 0 2 0 4mm 1 4 0 8mm 2 6 1 2mm 3 8 1 6mm 4 10 2 0mm 5 12 2 4mm 6 15 3 0 mm 7 20 4 0mm LEFT Digit Frequency Selector Digit Program Description 0 1 cycle Stretch 0 5 sec Hold indefinitely 1 cycle 1 Square wave pattem Stretch 0 5 sec Hold 1 sec Contract 0 5 sec End 1 cycle 2 Square wave pattern Stretch 0 5 sec Hold 3 sec Contract 0 5 sec End 1 cycle 3 Square wave patiem Stretch 0 5 sec Hold 10 sec Contract 0 5 sec End 1 cycles minute 4 Square wave pattem Stretch 0 5 sec Hold 29 5 sec Contract 0 5 sec Hold 29 5 sec Repeat 6 cycles minute 5 Square wave pattern Stretch 0 5 sec Hold 4 5 sec Contract 0 5 sec Hold 4 5 sec Repeat 20 cycles minute 6 Square wave pattem Stretch 0 5 sec Hold 1 sec Contract 0 5 sec Hold 1 sec Repeat 60 cycles minute 7 Square wave pattern Stretch 0 5 sec Contract 0 5 sec Repeat sec is the abbreviation for seconds 60 cycles minute is the maximum speed at which the instrument can operate At this speed the square wave pattern does not have a hold time Section 4 FAQ Q1 What are the characteristics of the silicone chamber A1 The strain chamber is made from silicone elastomer consisting of polydiethylsiloxane as its major component The chamber surface is strongly hydrophobic and cells have difficulty2. A STREX Cell Strain Instrument Cat ST 150 Serial 20B 00005 User Manual B Bridge International Inc 320 Logue Avenue Mountain View CA 94043 USA Phone 650 969 7727 Fax 650 969 7737 Email customersupportAb bridge com www b bridge com Contents Section 1 d ee e E 3 Ell Ee SOM ON ea eo Ne Et 3 Silicone Strain CC lune EE 3 Control Unt Fontanella a ea 4 Control UntBack Panel cien ments sea mass ade ted esa gia Deals 4 Section 2 Use of the Cell Strain Instrument een 5 Preparation of the Cell Strain Instrument ecscsecsssecesessesesseeeeeeeeseescaeseeeneeseueeeeeeeseeeeeeeeseaeeees 5 System Operation sss tat ates 5 Culturing Cell in the Silicone eene TEE 6 Preparation of Silicone Chambers ENEE 6 Section 3 Strain Parameters 1 2meiminaaiaimizamimstmiiimiimnnimsiiiamaisttitmi mim xim 8 SECHON A FAQ a eociineansiosammta sitaiatmicintmtmsimatisimeiomnndbrimagmsntaamnm mb mt bla 9 El UE 11 Section 6 Safety Jett Lema tada ba gEgge eEecgrEe Eege stat tacat 13 Section 7 Wairanty siisii usioe dadinne naand dodan oai ee danassut ccnansnadaucunsuadnaeatdedensbedsnsase dh Sa daaa aaea diaaa 15 Section 1 Main Components Cell Strain Instrument Stretch Unit Strain chamber brackets Each chamber is mounted on four brackets Fan Chamber Length Adjustment Knob To freely rotate the knob the power switch must be OFF Use the knob to adjust the distance of the chamber brackets such that it main
3. K Naruse Life Sci 69 15 1717 1724 2001 Uni axial cyclic stretch induces the activation of transcription factor nuclear factor KB in human fibroblast cells H Inoh N Ishiguro S Sawazaki H Amma M Miyazu H Iwata M Sokabe K Naruse FASB Journal 16 405 407 2002 Mechanical stress dependent secretion of interleukin 6 by endothelial cells after portal vein embolization Clinical and experimental studies M Kawai K Naruse S Komatsu S Kobayashi M Nagino Y Nimura M Sokabe J Hepatol 37 2 240 246 2002 Calcium regulates the P13K Akt pathway in stretched osteoblasts T Danciu R Adam K Naruse M Freeman P Hauschka FEBS Letters 536 193 197 2003 Anew mechanosensitive channel SAKCA and new MS channel blocker GSTMx 4 M Sokabe K Naruse T 11 18 19 20 21 22 23 24 25 Qiong Yao Folia Pharmacologica Japonica 124 3 301 310 2004 Mechanotransduction of integrin is essential for IL 6 secretion from endothelial cells in response to uniaxial continuous stretch A Sasamoto M Nagina S Kobayashi K Naruse Y Nimura M Sokabe Am J Physiol Cell Physiol 288 1012 1022 2005 N cadherin mediated cell adhesion determines the plasticity for cell alignment in response to mechanical stretch in cultured cardiomyocytes T Matsuda K Takahashi T Nariai T Ito T Takatani Y Jujio J Azuma Biochem Biophys Res Comm 326 228 232 2005 N cadherin signals through Rac1 determine the loc
4. Baggy clothing neckties necklaces etc can get tangled in moving parts of the unit Take appropriate precautions to prevent this occurrence Keep the machine clean and periodically inspect the instrument for excessive wear or damage Contact B Bridge if you have any concerns 14 Section 7 Warranty 1 The warranty is for one year commencing the date the customer receives the product and includes the instrument casing non wearable parts as well as the motor and bearings The cell culture chambers are considered consumables B Bridge International Inc is responsible for repair or replacement of chambers only if they are received and found defective 2 The warranty does not cover damage to the instrument that is a result of the following circumstances Damage caused by dropping or other impact Damage caused by inappropriate operation of the instrument Damage resulting from an attempted repair or modification of the instrument by the user Damage caused by unavoidable external causes such as earthquakes lightening fire flood gas leak power surges or other acts of providence O80 3 B Bridge International Inc is free from any responsibility for effects or loss or damages arising from the result of the machine operation This warranty assures that B Bridge International Inc will repair our product free of charge as stipulated in our warranty policy Any shipping charges will be born by buyer B Bridge Inte
5. cell scraper Q6 want to use recombinant cells for an experiment A6 Direct transfection of cells in the chamber may be possible However transfection itself may damage the cells which may make getting clear image data difficult We recommend performing the transfection in a standard culture dish then transferring the recombinant cells into the strain chamber Q7 Cells seem to be crowded in the center instead of being uniformly distributed throughout the chamber A7 Vibration from the incubator may disrupt the distribution of the cells We recommend gently rocking the chamber 15 mins after seeding your cells 10 Section 5 References 10 11 12 13 14 15 16 Effects of repetitive stretch stimulation on neonatal rat cardiocytes in vitro R Kada K Yasui K Naruse and J Toyama Environmental Medicine 40 69 72 1996 Inhibitory action of repeated stretch stimulation on apoptosis in neonatal rat cardiocytes K Yasui H Shimano K Kada K Naruse and J Toyama Environmental Medicine 40 175 177 1996 Mechanosensitive ion channels Single channels vs Whole cell activities M Sokabe K Nunogaki and K Naruse Progress in Cell Research 6 139 149 1997 Up regulation of integrin beta3 expression by cyclic stretch in human umbilical endothelial cells M Suzuki K Naruse Y Asano T Okamoto N Nishikimi T Sakurai Y Nimura and M Sokabe Biophys Biochem Res Com 239 372 376 1997 Mechano
6. alization of connexin 43 in cardiac myocytes T Matsuda Y Jujio t Nariai T Ito M Yamane T Takatani K Takahasi J Azuma J Mol Cell Cardio 40 4 495 502 2006 Activation of a mechanosensitive BK channel by membrane stress crated with amphipaths Mol Membr Biol 22 6 519 527 2005 Stretch induced cell proliferation is mediated by FAK MAPK pathway Life Sci 76 24 2817 2825 2005 Fabrication of reconfiguration protein matrices by cracking X Zhu K Mills P Peters J Bahng El Liu J Shim K Naruse M Csete M Thouless S Takayama Nature Materials 4 403 406 2005 Involvement of reactive oxygen species in cyclic stretch induced NF B activation in human fibroblast cells H Amma K Naruse N Ishiguro M Sokabe Brit J Pharmacol 145 364 373 2005 Viscoelastic and dynamic nonlinear properties of airway smooth muscle tissue roles of mechanical force and the cytoskeleton S Ito A Majumdar H Kume K Shimokata K Naruse K Lutchen d Stamenovic B Suki Am J Physiol Lung Cell Mol Physiol 290 6 L1227 1237 2006 Bi phasic activation of eNOS in response to uni axial cyclic stretch is mediated by differential mechanisms in BAECs H Takeda K Komori N Nishikimi Y Nimura M Sokabe K Naruse Life Sci 79 3 233 239 2006 12 Section 6 Safety Instructions Please read this section carefully before using the instrument Items in this section alert the user to operational dangers that if not followed may damage t
7. attaching to it therefore the chamber surface should be coated with an extra cellular matrix like fibronectin collagen laminin or gelatin before cultivation Q2 Cell attachment on the stretch chamber is not consistent A2 There may be wrinkles or bubbles on the bottom surface of the strain chamber when seeding cells Although the chamber is carefully made not to have wrinkles on it some products might have little wrinkles due to its thin structure We recommend the following steps Using a Petri dish that is large enough to hold the chamber add a small volume of ethanol Place one end of the chamber in the ethanol and lay the chamber down by slowly moving toward the opposite end of the chamber without trapping air bubbles between the dish and the chamber The thin layer of ethanol between the dish and the chamber will remove any wrinkles in the chamber membrane Allow the ethanol to evaporate before spreading your cell suspension in the chamber Q3 Cell attachment on the stretch chamber was confirmed by microscopy But the cells detached from the chamber surface after stretching the cells A3 Try seeding your chambers at a lower concentration of cells In typical cell culture dishes over confluent cells generally adhere to neighboring cells rather than to the base matrix dish surface This behavior is exaggerated when an excess number of cells are seeded into a stretch chamber Asecond possibility for cell detachment is that the cells
8. ature is excessively high Do not place and run the machine near a heater or in a place being exposed to direct sunlight To avoid possibly explosion never place and run the instrument nearby the presence of flammable solid substance liquid or gas It may cause explosion or fire Use the machine in well lit conditions Do not use the machine outdoors in direct sunlight or rain which may cause overheating or short circuit 13 Operational Concerns Please make sure to read the manual prior to running the unit Those who are not familiar with the machine should not operate it Do not put your hand close to mechanical parts or alike while the unit is running Do not put any foreign substances inside the machine Water metal or paper in motor area may cause fire or electrical shock Do not make any attempt to disassemble or modify the machine Do not remove the cover in an attempt to touch the mechanism inside which may cause you an electrical shock Please refrain from modifying the machine without our permission you may be shocked or injured If you do attempt to modify the machine the warranty on the unit is void and we will not be responsible for any performance deterioration or unit malfunction In the case of any abnormal sound smell or smoke disconnect the power immediately and contact B Bridge International Do not run the machine overloaded Be cautious as to your clothing and hair when operating the instrument
9. e This supplies electricity to the Stretch Unit and enables the two units to communicate 2 Connect the Control Unit to the power supply 240 V by the Power Cable 3 Turn on the Power Switch to start operation The Power Switch will light up and the Stop Button will flash BEWARE The Fan that cools the step motor will be rotating Do not obstruct the fan blades with any objects 4 If the electrical system is operating properly turn OFF the Power Switch 5 Make sure that the microscope is level on the work bench and the Stretch Unit when placed on the microscope is also level gt Start Cell Stretching 1 Make sure that the electrical communications for the Stretch and Control Units are properly set up 2 Load the chambers into the Stretch Unit by inserting the pins on the bracket into the four corners of the chamber The Power Switch must be OFF to freely rotate the Chamber Length Adjustment Knob 3 Rotate the Chamber Length Adjusting Knob counterclockwise to create tension on the chamber The membrane at the bottom of the chamber should be taut 4 Turn on the Power Switch 5 Select the desired stretch ratio and frequency on the Control Unit Programs are outlined in Section 3 6 Press the START button to start the stretch cycle During the action cycle the green light will be illuminated To STOP movement at any time press the STOP button 7 Do not change the Stretch Ratio or Frequency parameters during the opera
10. he instrument or more significantly result in serious injury or death of the user To ensure safe operation of the instrument it is therefore imperative that you follow these instructions carefully Power cable To avoid possible short circuit shock or fire Only use the power cable provided with the Cell Strain Instrument Do not touch the cable with wet hands Do not use the machine with other voltage than that specified In some cases a transformer may be used for compatibility Inappropriate current may result in the machine overheating short circuiting and or fire may occur Do not staple around the power cable Do not bend the cable or place heavy objects on it When pulling a connector from an outlet pull to disconnect gently by holding its plug not the cable Do not plug many objects into a single electrical outlet since it may cause fire If you are using an extension cord ensure it can withstand the total current to be used Disconnect power from the unit when it is not in use Connect the instrument to a power surge protected outlet Installation Location and Environment Keep the instrument on a stable level floor or a table secure from vibrations Be sure you have enough space Do not store the instrument in a humid or dusty place Over time excessive humidity or dust may cause deterioration that can result in an electrical short circuit and possibly fire Do not use the machine in a place where the temper
11. herefore the chamber must Coating with gelatin solution be coated with a call adhesion matrix 1 Pour 3 ml of the gelatin solution into each strain chamber 2 Incubate at 37 C for more than 30 minutes 3 Aspirate the gelatin solution If coating is successful water will not be repelled after removing the gelatin solution adhere to the matrix by integrins Integrin 4 The liquid solution can be used to coat 3 or 4 chambers before attachment is a cell specific interaction discarding unlike cell attachment to plastic or glass dishes where cells non specifically such as fibronectin or collagen In the presence of fibronectin or collagen cells gt Collagen Coating Cellmatrix 1 C P Type 3 or 4 attach due to a charger surface Preparation of collagen solution 1 Combine 1 part collagen to 10 parts HCL pH 3 in a sterile tube Coating with collagen solution 1 Coat chamber with a thin layer 2 Aspirate excess 3 Dry in biological safety cabinet at 25 or belo vv The chamber can be stored at the same temperature 4 Wash the chamber twice with culture medium 5 If coating is successful water will not be repelled Important If cells are having difficulty attaching to the freshly coated chambers or are easily detaching upon stretching treat the chamber with a higher concentration of the extra cellular matrix or coat overnight Section 3 Strain Parameters RIGHT Digit Stretch Ratio Selector
12. on matrix The coating procedures can be adapted for use with other matrices such as elastin pronectin and laminin Sterilize the chambers in an autoclave for 20 minutes at 121 C The silicone chambers can withstand temperatures up to 180 Use of an autoc lave is preferable However if an autoclave is not available the chambers may be sterilized by submerging them in 70 ethanol rinsing with water then drying in a sterile environment Place the sterile chambers in a Petri dish in preparation for coating gt Fibronectin Coating Preparation of fibronectin solution PBS per liter 1 Dilute human or bovine fibronectin to a final concentration of 50 to 100 ug ml in Phosphate Buffered Saline PBS NazHP4 anhyd KH2POs anhyd Coating with fibronectin solution 1 Pour 3 ml of the fibronectin solution into each strain chamber 2 Incubate at 37 C for more than 30 minutes 3 Aspirate the fibronectin solution If coating is successful water will not be repelled after removing the fibronectin solution 4 The liquid solution can be used to coat 3 or 4 chambers before discarding Note Dulbecco s PBS in powder form for tissue culture applications is also commercially available gt Gelatin Coating Preparation of gelatin solution 1 Add gelatin powder to PBS at a concentration of 2 The PDMS slicone chamber is very 2 Autoclave the mixture to dissolve and sterilize hydrophobic with two methytbases on the surface t
13. rnational Inc 320 Logue Avenue Mountain View CA 94043 USA Phone 650 969 7727 Fax 650 969 7737 Email customersupport b bridge com The information contained herein such as specification configuration and data or alike in part or in whole may be subject to change without notice 15
14. tains tension on the silicone chamber Motor Cable Cable connects to Control Unit Platform Place platform on top of microscope stage for viewing Silicone Strain Chamber ST CH 04 0 Chamber Dimensions 20mm x 20mm x 10mm Chamber membrane thickness 0 1mm 4 places for pins Control Unit Front Panel Main Power Switch PATTERN Start and Stop Button Use to start or stop the stretching action Frequency Selector left digit Stretch Ratio Selector right digit Use upper and lower buttons to adjust frequency Use upper and lower buttons to increase and decrease strain ratio Control Unit Back Panel Power Cable Outlet Fuse Motor Cable Outlet Connects the Stretch Unit to the Control Unit via Motor Cable Section 2 Use of the Cell Strain Instrument Preparation of the Cell Strain Instrument Before using the Stretch Unit sterilize the unit especially the chamber mounting area using ethanol immersed swabs System Operation Operation of the Cell Strain Instrument is very straight forward and intuitive Below are the basic steps to use this instrument The step motor that moves the chamber brackets is made to operate 15 minutes continuously It is not recommended to extend the use of the motor beyond 15 continuous minutes because of possibly overheating and burning out the motor gt Set up the Stretch Unit and Control Unit 1 Connect the Stretch Unit to the Control Unit by the Motor Cabl
15. tion of a stretch cycle Press the STOP button and wait for the program to complete the final stretch contract sequence returning to the original start position before initiating the next stretch parameter Changing parameters during a stretch cycle may damage the motor 8 After a few minutes of running a stretch program stop the cycle and check the condition of the cells If the cells have not detached from the membrane proceed with your experiment If the cells are detached the chamber coating was probably insufficient Recoat the chambers Culturing Cells in the Silicone Chambers 1 Seed cells at the appropriate concentration in a freshly coated chamber Important It is critical to not over expose the cells to dissociation enzymes Cells should be treated in the same manner type and concentration of enzyme temperature and exposure time for all experiments Important Cells should not be seeded at a high density in the chambers For example epithelial cells often form a cell sheet and the cell cell adhesion seems to be stronger than a cell surface adhesion When this happens cells may detach from the chamber Additionally cultures that are grown over a week in the chambers may detach 2 After an overnight incubation without stretching inspect the cells with a microscope to ensure they have adhered to the chamber Preparation of Silicone Chambers Before using the chambers they should be sterilized and coated with a cell adhesi
16. transduction and intracellular signaling mechanisms of stretch induced remodeling in endothelial cells Masahiro Sokabe Keiji Naruse Shorei Sai Takako Yamada Keisuke Kawakami Masumi Inoue Kichiro Murase and Motoi Miyazu Heart Vessel S12 191 198 1997 Involvement of SA channels in orienting response of cultured endothelial cells to cyclic stretch K Naruse Y Yamada and M Sokabe Am J Physiol 274 H1532 H1538 1998 Up regulation of COX expression by uni axial cyclic stretch in human lung fibroblast cells T Kato N Ishiguro H Iwata T Kojima T Ito and K Naruse Biophys Biochem Res Com 244 615 619 1998 Pp125 is required for stretch dependent morphological response of endothelial cells K Naruse T Yamada X Sai M Hamaguchi and M Sokabe Oncogene 17 455 463 1998 Orientation Change of Cardiocytes Induced by Cyclic Stretch Stimulation Time Dependency and Involvement of Protein Kinases K Kada K Yasui K Naruse and J Toyama J Mol Cell Cardio 31 247 259 1999 Molecular Identification of a Eukaryotic Stretch Activated Nonselective Cation Channel M Kanzaki M Nagasawa L Kojima C Sato K Naruse M Sokabe H lida Science 285 882 886 1999 Activation of pp60S is Critical for Stretch Induced Orienting Response in Fibroblasts X Sai K Naruse M Sokabe J Cell Sci 12 1365 1373 1999 SA Channel Mediates Superoxide Production in HUVECs K Aikawa N Nishikimi T Sakurai Y Nimura M Sokabe
17. were damaged by enzyme treatment such as trypsin The damaged cells may attach to surfaces by non specific binding and are not specifically bound to the extra cellular matrix coating on the chamber therefore time concentration and temperature for the enzyme treatment should be optimized to reduce cell damage A third possibility is insufficient coating of the chamber preventing the cells from attaching to the chamber In this case longer coating time is recommended Some researchers coat the chamber with two or more kinds of the extra cellular matrix materials to increase binding effectiveness Q4 How long can cells be stretched A4 The duration depends on cell strain and condition However the step motor in a ST 150 and ST 195 is made to operate for 15 continuous minutes The ST 140 models can run for hours to days if the cooling system is on Q5 How can obtain protein or mRNA samples from the cells attached to the silicone membrane A5 1 Proteins for Western blotting Wash the cells once with PBS Add SDS PAGE sample loading dye directly into the chamber and collect the cell extract by using a cell scraper 2 Proteins for Immunoprecipitation Wash the cells once with PBS Add cell extract buffer directly into the chamber and collect the cell extract by using a cell scraper 3 RNA Wash the cells once with PBS for RNA preparation Add RNA extraction buffer directly into the chamber and collect the cell extract by using a
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