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STREX Cell Strain Instrument User Manual
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1. A STREX Cell Strain Instrument Catalog ST 140 04 Serial 20A 00013 User Manual B Bridge International Inc 320 Logue Avenue Mountain View CA 94043 USA Phone 650 969 7727 Fax 650 969 7737 Email customersupport b bridge com www b bridge com Contents Section 1 Main ComponentsS rerrssrnarnansnnnnannnnnnnnnnnennnnansnnennnnnnennnnnnsnnnnnnnnnennnsnnnnnnnnnennnnnnsnnnnnnnnnnennnnen 3 LG APEE EEE EAEE E E E EAE EEE E E EE EEEE EE E 3 ETO ME OPA aran as dads aa tan as decades ane eines nanan acne 4 CONVO BICK PAN sva 4 Electrical Transformer amp Electrical Assembly c cccccceccessessessessessecsessesseeseeseeseessesaesaeesesaeeaeeaes 5 SlliCone STAN Chamber var 6 Section 2 Use of the Cell Strain Instrument rnvrnnrnnrnnrnnvnnvennnnnvnnnnnnnnvnnvnnennnnnnnnnnnnnnnnnnnnnnnnennennenn 7 Preparation of Cell Strain Instrument wrvennernronrernrrnvenrernvrnvennennvenvennnsnvennennvenvennnsnvennennvsnvennsnnen T SEN OPSA ON naa T 7 Culturing Cells in the Silicone Chambers araarvarvanrrnrranrnnrrnnrnnrnar e eee eeeeeeeaeeaeeaaes 9 Preparation of Silicone OED ES ae ode 9 Section 3 Strain Parameters rnrnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnunnnnnnnnnnnneuunnuenuunnunnnnnn 11 SECTION 4 FAQ save 12 SECUON DS ReETEPENCES iniinum a 14 Section 6 Safety Instructions 2 asia 16 Section 7 VV AN AIM 2 18 Section 1 Main Components Stretch Unit Chamber length adjusting k
2. may damage the cells which may make getting clear image data difficult We recommend performing the transfection in a normal culture dish then transferring the recombinant cells into the strain chamber Q7 Cells seem to be crowded in the center of the chamber instead of being uniformly distributed throughout the chamber AT Vibration from the incubator may disrupt the distribution of the cells We recommend gently rocking the chamber 15 minutes after seeding your cells 13 Section 5 References 10 11 12 13 14 15 16 Effects of repetitive stretch stimulation on neonatal rat cardiocytes in vitro K 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 Mechanotransduction and intracellular signaling mechanisms of stretch induced remodeling in endothelial cells Masahiro Sokabe Keiji Naruse Shorei Sai
3. 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 kB 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 Komoni N Nishikimi Y Nimura M Sokabe K Naruse Life Sci 79 3 233 239 2006 15 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 the instrument or more significantly result in serious injury or death of the user To ensure safe operation of the instrument it is
4. Takako Yamada Keisuke Kawakami Masumi Inoue Kichiro Murase and Motoi Miyazu Heart Vessel 512 191 193 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 Kojima C Sato K Naruse M Sokabe H lida Science 285 882 886 1999 Activation of pp60 gt 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 K Naruse Life Sci 69 15 1717 1724 2001 Uni axial cyclic stretch induces the activation of transcription factor nuclear factor KB in h
5. h cold water Use the Power Cable to connect the Control Unit to an electrical transformer Electrical Transformer AG ANTMATIG PAADIST EU amp MODEL SVC 600ND I OUTPUT mi lear thar Electrical Transformer Also known as Automatic Voltage Regulator Electrical Assembly of STREX Back panel of Control Unit Stretch Unit Transformer i oe er ab weer syl fied ode FLL VL LER ar a na haa a oa A A aa Plug Adapte STREX Power Cable 230 volt Power source Connecter Cable Silicone Strain Chamber ST CH 04 0 Chamber Dimensions 20mm x 20mm x 10mm 31m EN Chamber membrane thickness 0 1mm 4 places for pins 40m Section 2 Use of the Cell Strain Instrument Preparation of the Cell Strain Instrument Before using the Cell Strain Instrument sterilize the unit especially the chamber mounting area using ethanol immersed swabs System Operation The STREX Cell Strain System needs to be water cooled when in operation Without cooling the maximum run time for the continuous operation of the motor is only 15 minutes For longer operation water cooling is essential Also please note that when operating the unit in an incubator the temperature inside the unit will be elevated so it is particularly important to ensure the cooling system is employed in this environment gt Set up the Strain and Control Unit 1 Set up the cooling system by usi
6. his occurrence Keep the machine clean and periodically inspect the instrument for excessive wear or damage Contact B Bridge if you have any concerns 17 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 ONO 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 International Inc 320 Logue Avenue Mountain View CA 94043 USA Phone 650 969 7727 Fax 650 969 7737 email customersupport b b
7. id 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 16 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 Baggy clothing neckties necklaces etc can get tangled in moving parts of the unit Take appropriate precautions to prevent t
8. ls 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 Place a small volume of ethanol in a Petri dish that is large enough to hold the strain chamber Gently place the chamber in the culture dish starting at one edge and moving toward the opposite edge of the chamber to remove air bubbles between the dish and chamber 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 Over confluent cells generally adhere to neighboring cells rather than to the base matrix dish surface When an excess amount of cells are put in a culture dish the cells connect to each other after growth Such behavior of the over confluent cells which are often observed in normal culture dishes is even worse in the strain chamber Asecond possibility for cell detachment is that the cells were damaged by enzyme treatment such as trypsin before seeding The damaged cells sometime 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 thi
9. ng the Connector Cable The Connector Cable supplies electricity and cold water from the Control Unit to the Stretch Unit One end of the Cable has 3 plugs of which 2 are identical metal plugs This end of the Cable attaches to the front of the Stretch Unit The 2 identical metal plugs are plugged into the System Coolant Inlet and Outlet Valves see Section 1 To release the metal plugs from the Stretch Unit push backward on the outer metal rings around the valves The single larger black plug supplies electricity to the Stretch Unit this plug has to be in the correct orientation to fit properly Improper orientation will break the outlet The other end of the Connector Cable has 2 exposed latex tubes which fit onto the System Coolant Inlet and Outlet Valves on the back panel of the Control Unit The Cable s square shaped plug fits the motor outlet just above the coolant valves To circulate cool water throughout the system use latex tubing to attach a large reservoir of cold water to the Reservoir Coolant Inlet and Outlet Valves on the back panel of the Control Unit The water in the reservoir can be kept cool by placing the reservoir on ice 2 The Control Unit was made for 110 Volts therefore the Control Unit must be connected to an electrical transformer refer to section 1 by the Power Cable 3 Please read the Stavol instructions before using the transformer A Use the plug adapter to connect the transformer to your 230 Volt powe
10. nob Use the knob to adjust the distance of chamber brackets to maintain tension on the chamber The bottom of the chamber should be taut System coolant inlet and outlet valves Be sure to supply cool water when in operation Failure to do so may lead to overheating inside the incubator or motor burnout Connectors Use Connector Cable to connect Control Unit to Stretch Unit Strain Chamber Brackets Each chamber is mounted on four pins two pins on each bracket Mounting Stage Brackets sit on top of the stage Alignment Pins Holds Strain Chamber Brackets in place Control Unit Front Panel Start and Stop Button Use to start or stop the stretching action ER p Main Power Switch sc ao m _ IT Water Pump Switch an Use to tum on and off the pump that circulates water to cool the motor Strain Ratio Use upper and lower buttons to increase and decrease stretch ratio Strain Frequency Selector Use upper and lower buttons to adjust cycle frequency Control Unit Back Panel Control Connector This cable supplies electricity from the Control Unit to the Stretch Unit System coolant inlet amp outlet valves Be sure to supply cool water when in operation Failure to do so may lead to overheating inside the incubator or motor burnout Fuse case cover Reservoir coolant inlet amp outlet valves Power Cable Outlet for 110 Volts Attach a large flask or similar type of reservoir filled wit
11. r source B Flip the transformer s Input Switch to the ON position C When the orange light is ON and the volt meter indicates 100 Volts then the transformer is ready to use D Connect the STREX Control Unit to the transformer using the Power Cable The Power Cable connects from the back of the Control Unit to the Output 8 plugs in the transformer E Flip the transformer s Output Switch to the ON position IMPORTANT When turning OFF the STREX instrument follow the steps in the order listed below Tum OFF the STREX Control Unit 2 Flip the Output Switch to the OFF position 8 Flip the Input Switch to the OFF position Turn on the Main Power Switch and Water Pump Switch on the Control Unit The Switches will light up Press the START button to ensure that the strain chamber bracket moves correctly The Start Button light will flash when the instrument is stretching Press the STOP button to ensure that the strain chamber bracket stops moving The Stop Button will light up Make sure that the water pump Is pumping water from the reservoir and through the Control and Stretch Unit Turn off Main Power and Water Pump Switches Start Cell Stretching 1 The Power Switch must be OFF to freely rotate the Chamber Length Adjustment Knob Silicone chambers containing cultured cells are attached to the Stretch Unit by inserting the 4 pins into the corners of the chamber Rotate the Adjustment Knob clockwise until the bo
12. rd possibility is insufficient coating of the chamber preventing the cells from attaching to the chamber Longer coating time is recommended Some researchers coated the chamber with two or more kinds of the extra cellular matrix materials to increase binding effectiveness 12 Q4 How long can cells be stretched A4 The duration depends on cell strain and condition In general two weeks cultivation in the incubator is possible with culture media changes every 2 3 days It is also important to monitor reservoir coolant for the instrument motor Insufficient amount of the cooling water may increase the temperature of the motor resulting in killing of cells and or burning out the motor Q5 How can I obtain protein or mRNA samples from the cells attached to the silicone membrane Ao 1 Proteins for Wester 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 cell scraper Q6 want to use recombinant cells for an experiment AG Direct transfection of cells in the strain chamber may be possible However transfection itself
13. retch RIGHT Digit Strain Ratio Ts 2 15 10m 3 8 16m 4 10 20m 8 15 30mm 6 16 32mm LEFT Digit Strain Frequency Selector ERE ae mete tt Square wave Hold 49 sec stretch 1 sec hold 49 sec contract 1 sec repeat a o Saame Seg 1 Square wave Hold 29 sec stretch 1 sec hold 29 sec contract 1 sec repeat a Sene Holi 2soc steirt each eee contact ec ropest 2 Square wave Hold 2 sec stretch 1 sec hold 2 sec contract 1 sec repeat Po Pape memene O 3 Sine wave Stretch 1 sec contract 1 sec repeat ce Pam nosa oaos 4 Sine wave Stretch 0 5 sec contract 0 5 sec repeat Stretch 1 sec hold 3 hours contract 1 sec 6 Static Stretch 1 sec hold indefinitely Stretch 0 5 sec hold indefinitely sec is the abbreviation for seconds 11 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 difficulty 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 cel
14. ridge 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 18
15. t cells under a microscope to ensure that they adhered to the chamber Preparation of Silicone Chambers Before using the chambers they should be sterilized then coated with a cell adhesion matrix The coating procedures below can be adapted for use with other matrices such as elastin pronectin and laminin Sterilize chambers in an autoclave for 20 minutes at 121 C The silicone chambers can withstand temperatures up to 180 C Use of an autoclave is preferable However if an autoclave is not available the chambers may be sterilized by submerging in 70 ethanol rinsing with water then drying in a sterile environment Place the sterile silicone chambers in a Petri dish in preparation for coating Fibronectin Coating Preparation of fibronectin solution NaCl ne 00 1 Dilute human or bovine fibronectin to a final concentration of 50 020 to 100 ug ml in Phosphate Buffered Saline PBS NazHPa anhyo L159 KH2P Oa anhyd 0 209 Coating with fibronectin solution 1 Pour 3 ml of the fibronectin solution into each strain chamber Note Dulbecco s PBS in powder form for 2 Incubate at 37 C for more than 30 minutes peker pea 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 Gelatin Coating Preparation of gelatin solution 1 Add gelatin powder to PBS at a concen
16. 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 leveled 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 temperature is excessively high Do not place and run the machine near a heater or in a place being exposed to a direct sunlight To avo
17. tration of 2 2 Autoclave the mixture to dissolve and sterilize Coating with gelatin solution 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 4 The liquid solution can be used to coat 3 or 4 chambers before discarding gt Collagen Coating Cellmatrix 1 C P Type 3 or 4 The PDMS Silicone chambers are very hydrophobic with two methyl bases on Preparation of collagen solution the surface Cells adhere to the 1 Combine 1 part collagen to 10 parts HCL pH 3 in a sterile fibronectin or collagen coated chambers tube via integrins This form of cell adhesion is very different compared to attachment of Coating with collagen solution cells to plastic or glass dishes where the 1 Coat chamber with a thin layer surface of plastic or glass is charged 2 Aspirate EXCESS resulting in non specific binding 3 Dry in biological safety cabinet at 25 C or below 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 detached upon stretching treatthe stretch chamber with a higher concentration of fibronectin or collagen Ifthe cells are having difficulty attaching to the coated chamber or easily for ovemight 10 Section 3 Strain Parameters St
18. ttom of the chamber is taut Select the stretch parameters by changing the Stretch Pattern Buttons Refer to Section 3 for stretch parameters Do not change the Strain Ratio or Strain Frequency Selector Buttons during the operation of a stretch cycle Press the Stop Button and wait for the last cycle to complete and the chamber returns to its starting position before changing stretch parameters Changing parameters during a stretch cycle may damage the motors Push the Start Button to start the stretch unit After 5 minutes stop the stretching and check the condition of the cells If the cells have not detached proceed with your experiment If the cells are detached the adhesion matrix coating was probably insufficient Recoat the chambers Culturing Cells in the Silicone Chambers 1 Seed cells at the appropriate concentrations in the freshly coated chambers Important It is critical to not over expose the cells to dissociation enzymes Cells should be treated in the same manner type concentration of enzyme temperature and exposure time for all experiments Important Cells should not be cultured at a high cell 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 overnight incubation inspec
19. uman 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 Gs TMx 4 M Sokabe K Naruse T 14 17 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 localization of connexin 43 in cardiac myocytes T Matsuda Y Jujio t Nariai T Ito M Yamane T Takatani K Takahasi J Azuma J
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