Micro-Incubation Chamber for 35 mm Cell Culture Dishes Model DH
Contents
1. O2 The Middle Section and windowed Top Section are easily removed for replacement of cell culture dishes during replicate trials The Middle section locks onto the Bottom Section holding the cell culture dish via the two magnetic posts Dish clamps can still be used in the closed format via the slots provided in the Middle Section to insure good thermal contact between the cell culture dish and the resistive heating elements A special hole in the Middle 12 Section permits insertion of out T2 bath thermistor in the enclosed cell culture dish In the closed format resistive heating can be achieved in the same manner as described above for the open format The closed format permits imaging using inverted microscopes 7 Warner Instruments Inc DH 35i Rev 05 07 03 13 APPENDIX Cleaning and maintenance The DH 35i is constructed of anodized aluminum It can be easily cleaned with common laboratory detergents or with ethyl alcohol Warranty and service Service We recommend that all questions regarding service be referred to our Technical support Department Normal business hours are 8 30 AM to 5 00 PM EST Monday through Friday Our offices are located at 1125 Dixwell Avenue Hamden CT 06514 We can be reached by phone at 800 599 4203 or 203 776 0664 Our fax number is 203 776 1278 E mail us at support warneronline com or through the web at http www warneronline com contact html Warranty The2. CC 28 cable Heating of perfusate can be achieved using our SH 27B or SF 28 In Line solution heaters which are also controlled via our TC 324B and TC 344B Temperature Controllers Warner Instruments RC 37 perfusion chamber inserts e g RC 37W RC 37F are also available for Willco Corning and Falcon 35 mm cell culture dishes and can be used together with the DH 35i These incorporate our diamond fluidics design to maintain laminar perfusate flow Fluid level in the cell culture dish can be controlled via a height adjustable aspirator Agar bridge wells are built into the RC 37 perfusion chamber inserts for signal grounding purposes The combination of the RC 37 perfusion chamber inserts with our DH 35i micro incubation heater creates the ability to quickly alter the extracellular environment in 35 mm cell culture dishes by changing either the perfusate or the bath temperature perfusion aspiration port Closed Format Applications JE In the closed format the Middle m thermisitor input port Section and windowed Top Section are used together with the Bottom Section described above to maintain an Ve perfusion input port enclosed environment Gas inlet and T1 thermisitor outlet ports are available in the Middle Section for atmospheric control of the gas input port cell culture dish s environment This dish clamp enables the user to control the cell s 7 Warner Instruments Inc DH 35i Rev 05 07 03 pH CO2 and
3. DH 35i is covered by our Warranty to be free from defects in materials and workmanship for a period of one year from the date of shipment If a failure occurs within this period we will either repair or replace the faulty component s This warranty does not cover failure or damage caused by physical abuse In the event that repairs are necessary shipping charges to the factory are the customer s responsibility Return charges will be paid by Warner Instruments 7 Warner Instruments Inc
4. esM ren dd 13 Marranti and SeEVICO iii ai raa ETA EA 13 a 13 WTA Vs HA 13 Warner Instruments Inc DH 35i Rev 05 07 03 3 The DH 35i Micro Incubation Chamber from Warner Instruments is designed to provide complete environmental control over 35 mm culture dishes while maintaining the ability to make both short and long term imaging studies This versatile system readily accommodates culture dishes from several manufacturers and will adapt to any microscope stage supported by Warner Instruments Features of the DH 35i include v Designed for glass bottomed 35 mm cell culture dishes Permits imaging temperature and gas atmosphere control Adapts to Willco Wells and Corning and Falcon dishes Unique dish clamps permit either open or closed use x 08 Ss X Works with RC 37 family of Cell Culture Dish Perfusion Chamber Inserts Warner Instruments Inc DH 35i Rev 05 07 03 4 INTRODUCTION The introduction of affordable 35 mm cell culture dishes incorporating high quality glass bottoms has extended their use into areas such as confocal microscopy fluorescence microscopy and high resolution image analysis The DH 35i Culture Dish Incubator is designed to facilitate the imaging of live cells in a cell culture dish using either an open or closed dish format The open dish format readily accommodates the use of microelectrodes or rapid throughput tools such as the RC 37 Cell Culture Dish Per
5. DH 35i Rev 05 07 03 Warner Instruments Inc Micro Incubation Chamber For 35 mm Cell Culture Dishes Model DH 35i Warner Instruments Inc 1125 Dixwell Avenue Hamden CT 06514 800 599 4203 203 776 0664 203 776 1278 fax Warner Instruments Inc DH 35i Rev 05 07 03 Table of Contents DESERIPTION ia i a i A E A E 3 INTLRODUC IO Ni A 4 COMPONENTS uiis EHE RID ADM a enm EE 5 Bottoni SCC O 5 Middle CC CIO EIE TEST 5 Top L M 5 ACCESS ONTE O e A A A H 6 SETUP A 6 General UII PTT OT TO LN A 6 Open OPIN A E a A 6 Open Format Setup without PeP aad gyae Rar inkara aaa hE ET NESE EA ONAE NTE rE iari 6 Open Format Setup WUN TETE sessi Tai E EEE OIE EASIER TAER SS Ne BUTS aE Taita Closed EL m 8 Closed Format Setup without PCrfUsion iccsssescvcecssivacesteiassecaviasavssedstnussedcistntelstuacavinasiateedevesielaveseteddeisees 8 Em D a RE eC one ene POET eee tt a Closed Format Setup with Perfusloh T 9 OFERA TION e 11 Open Format Use c PA 11 Closed Format A ppliCaviOns si ssssccccvscasccccenssscsvssesescecsscancesnescecenseussterssseccecsusssevessnecssunsssstviseneecessassesessexs 11 APP END p C i 13 Teaming EST C T oieeedvovieeex vi deep al dei dA S Poss d o cta ve pd
6. apter rings from the Bottom Section Color codes for the different adapter rings are shown below Adapter ring color codes Willco Wells Models D3512 and D3522 Red o Falcon 37 mm Gold Falcon 38 mm Corning 25000 series 2 Special clamps two sets are provided to hold cell culture dishes in place in the Bottom section These clamps are shown in use in the top photo on page 5 Clamping insures good thermal contact between the culture dish and the heated surface which helps to maintain tight temperature control SETUP General Comments The DH 35i is assembled in stages from the bottom up The Bottom Section may be used alone for open format applications while additional assembly of the Middle and Top Sections are required for closed format applications The Middle Section contains several ports allowing gas line perfusion line electrode and thermistor access While the Middle Section has been designed for closed format applications it can also be used as a multi port holder for open format applications We recommend the use of the RC 37 Culture Dish Perfusion Insert when using the DH 35i in closed format Open Format Open Format Setup without Perfusion 1 Begin by selecting the appropriate adapter ring for your cell culture dish See adapter guide above 7 Warner Instruments Inc DH 35i Rev 05 07 03 7 2 Place the adapter ring notch side down on a firm surface and insert the cell c
7. ble tubing for the perfusion inlet port typically aligns itself in its slot with no difficulty and you can pull out any slack from inside the middle portion of the DH 35i The flexible tubing for the suction port typically requires a small degree of looping to enable it to both fit in its slot and to prevent kinking of the suction tubing See figure to the right Re adjust the height of the suction tubing via its Teflon component Place the Top Section of the DH 35i on top of the Middle Section There is an aligning hole on the top of the Middle Section and a matching pin on the bottom of the Top Section to facilitate proper alignment There are also three additional pins on the bottom of the Top Section which fit around the outside edges of the Middle Section If heating with Warner s TC 324B or TC 344B Temperature Controllers slip the white colored T1 feedback thermistor on the CC 28 cable into the milled hole in the side of the Bottom Section A small amount of mineral oil can facilitate thermal contact between the T1 feedback thermistor and the metal of the Bottom Section Slip the blued colored leads over the two resistive heating elements and then plug the CC 28 cable into the temperature controller Consult the TC 324B TC 344B user s manual for methods of controlling the temperature of the clamped culture dish If imaging place the fully assembled DH 35i into your microscope s Series 20 Stage Adapter a
8. en Format Setup with Perfusion 1 Perform steps 1 through 3 described in the Open Format Setup without Perfusion portion of this manual See page 6 2 Align the Bottom Section with the already clamped dish in place so that the white dot on the side of the Bottom Section faces towards the left i e roughly 9 00 The direction of the clamps holding the dish should be horizontal from left to right 7 Warner Instruments Inc DH 35i Rev 05 07 03 8 Placing and Clamping the RC 37 RC 37 perfusion inserts e g RC 37F RC 37W have both a perfusate inlet tubing port and a suction tubing port with a white Teflon height adjusting component These ports are each guarded by raised sections of the insert itself 3 With the perfusion inlet tubing port directed towards you i e roughly 6 00 slip the RC 37 between the clips and into the clamped dish The non raised sections of the RC 37 should easily slip under the clamps holding the dish When properly installed the white Teflon height adjusting component should be near the top of the clamped 35 mm dish i e roughly 12 00 4 Rotate the RC 37 clockwise until the raised sections of the insert bump up against the clamps The suction tubing port with its white Teflon component will be towards the right o While holding the RC 37 with your left hand further rotate the insert clockwise while simultaneously lifting the right side clamp use the right hand onto the raised sectio
9. fusion Chamber while the closed dish format can be used to perform long term studies or to maintain pH CO2 or other environmental factors Temperature control is available in both open and closed formats DH 35i incubators are designed to work with Willco Wells Corning or Falcon 35 mm glass bottom cell culture dishes The incubator is machined in three separate interlocking sections and metal adapters are supplied to insure a secure fit for supported cell culture dishes 7 Warner Instruments Inc DH 35i Rev 05 07 03 o COMPONENTS The completed DH 35i assembly is comprised of a bottom middle and top section along with culture dish adapter rings and an optional RC 37 chamber insert The completed assembly can be mounted onto a microscope using an appropriate microscope stage adapter and thermal control is provided using a CC 28 cable and temperature controller Bottom Section The Bottom Section positions the cell culture dish using one of four adapter rings see table on next page The bottom section also includes two resistive heating elements a 19 mm diameter aperture two magnetic posts for attachment of the Middle Section component two holes for the special dish clamps and a single hole for a attachment of a control thermistor e g via Warner s CC 28 cable The periphery of the bottom section is framed with stainless steel plates for mounting of magnetic holders when the DH 35i is used on the open co
10. n which guards the suction tubing port 6 Next hold down the RC 37 with the right hand and place the left side clamp on top of the raised section which guards the inlet tubing port 7 Center the diamond shaped chamber of the RC 37 insert on the window of the 35 mm cell culture dish and adjust the height of the suction tubing port via its white Teflon component 8 If heating perform step 4 described in the Open Format Setup without Perfusion portion of this manual see page 7 Closed Format Closed Format Setup without Perfusion la Begin by selecting the appropriate adapter ring for your cell culture dish See adapter guide above Place the adapter ring notch side down on a firm surface and insert the cell culture dish into the ring by gently pushing it into place IN Clamping The dish ring assemblv is placed into the recess in the Bottom Section and clamped into place Cell culture dish clamps 2 ea are provided and are placed into two small holes 7 Warner Instruments Inc DH 35i Rev 05 07 03 9 located on either side of the Bottom Section These holes can be found directly between the magnet and heating resister and can be located by a red colored dot on top of the rim on the Bottom Section Clamps are placed on both sides of the dish See the figure to the right for clamp positioning While holding down the dish with one hand slide the clamps into position with the other hand until they sec
11. nd mount onto the microscope 7 Warner Instruments Inc DH 35i Rev 05 07 03 11 OPERATION Open Format Applications Only the Bottom Section is required for open format use together with one of the four colored adapter rings and one set of dish clamps The open format enables easy access for microelectrodes as well as for perfusion and aspiration lines Cell culture dishes can be used with and without covers and dish perfusion can be simplified by use of Warner s RC 37 Perfusion Chamber Insert The open format permits imaging using either upright or inverted microscopes Warner Series 20 stage adapters are used to mount the DH 35i onto most commonly used microscope stages Contact our Tech Support Department for custom stage adapters Resistive heating of the DH 35i is achieved using Warner s TC 324B or TC 344B Temperature Controller and a CC 28 cable The two blue CC 28 connectors attach directly to each resistive heating element Feedback control is provided thermistor i e T1 is inserted in the Bottom Section s metal chassis via a special hole see photo We recommend using a drop of mineral oil to insure good thermal contact between the white T1 thermistor and the metal chassis The CC 28 cable s bath thermistor i e T2 can be placed directed in the bath to check solution temperature The temperature control unit regulates the heating current by sensing the temperature of the incubator via the T1 control thermistor in the
12. nfiguration Middle Section The Middle Section attaches to the Bottom Section via two magnetic points mounted on the Bottom Section The Middle Section has slots around its circumference to allow access for cabling and tubing elements Two recessed magnetic anchors couple directly with the Bottom Section Several slots are provided for access of perfusate inlet and outlet lines gas inlet and outlet lines and a hole for placement of a bath thermistor e g via Warner s CC 28 cable The small chrome tube shown in the picture above can be used to supply a slight vacuum to the chamber which will keep the top cover clear of condensation while the DH 35i is in use Top Section The Top Section rests on top of the Middle Section and forms an enclosed micro 7 Warner Instruments Inc DH 35i Rev 05 07 03 6 incubation system Alignment is achieved via is via three metal posts and an alignment pin The top section holds a 40 mm cover glass via a recessed annular lip and the cover glass is secured using a rubber o ring When in place the cover glass seals a 36 mm widow useful for illumination purposes Accessories 1 Four colored metal adapter rings are included to insure a close fit between the Bottom Section and the cell culture dish Rings are tapered for a precise fit and should be mounted with the notch facing down A special tool i e a modified 18G hypodermic needle is provided to assist in removing ad
13. ulture dish into the ring by gently pushing it into place IN Clamping The dish ring assemblv is placed into the recess in the Bottom Section and clamped into place Cell culture dish clamps 2 ea are provided and are placed into two small holes located on either side of the Bottom Section These holes can be found directly between the magnet and heating resister and can be located by a red colored dot on top of the rim on the Bottom Section Clamps are placed on both sides of the dish See the figure to the right for clamp positioning 3 While holding down the dish with one hand slide the clamps into position with the other hand until they secure the culture dish into place Verify that the dish is seated completely flat 4 If heating with Warner s TC 324B or TC 344B Temperature Controllers slip the white colored T1 feedback thermister on the CC 28 cable into the milled hole in the side of the Bottom Section A small amount of mineral oil can facilitate thermal contact between the T1 feedback thermister and the metal of the Bottom Section Slip the blued colored leads over the two resistive heating elements and then plug the CC 28 cable into the temperature controller Consult the TC 324B TC 344B user s manual for methods of controlling the temperature of the clamped culture dish o If imaging place the fully assembled DH 35i into your microscope s Series 20 Stage Adapter and mount onto the microscope Op
14. ure the culture dish into place Verify that the dish is seated completely flat Place the Middle Section of the DH 35i on top of the Bottom Section so that the two white dots on the side are aligned There are two long narrow slots for both clamps and two short wide slots for both resisters Place the Top Section of the DH 35i on top of the Middle Section There is an aligning hole on the top of the Middle Section and a matching pin on the bottom of the Top Section to facilitate proper alignment There are also three additional pins on the bottom of the Top Section which fit around the outside edges of the Middle Section If heating with Warner s TC 324B or TC 344B Temperature Controllers slip the white colored T1 feedback thermistor on the CC 28 cable into the milled hole in the side of the Bottom Section A small amount of mineral oil can facilitate thermal contact between the T1 feedback thermistor and the metal of the Bottom Section Slip the blued colored leads over the two resistive heating elements and then plug the CC 28 cable into the temperature controller Consult the TC 324B TC 344B user s manual for methods of controlling the temperature of the clamped culture dish If imaging place the fully assembled DH 35i into your microscope s Series 20 Stage Adapter and mount onto the microscope Closed Format Setup with Perfusion l 2 3 4 Perform steps 1 through 3 described above in the Closed Format Setup witho
15. ut Perfusion portion of this manual See page 8 Attach C Flex tubing to the output port and PE 50 tubing to the input port on your RC 37 Insert Align the assembled DH 35i so that the white dot on the side of the Bottom Section faces towards your left i e roughly 9 00 While holding the Middle Section of the DH 35i with its white dot also facing towards your left slip a portion of the RC 37 suction tubing through its matching slot on the right side of the Middle Section i e roughly at 2 00 Give yourself excess flexible tubing inside the Middle Section at this time 7 Warner Instruments Inc DH 35i Rev 05 07 03 10 2 10 11 12 Place the narrow slot and wide slot on the right side of the Middle Section on top of the matching right side clamp and resister respectively i e roughly 3 00 of the Bottom Section Slip a portion of the PE 50 tubing attached to the perfusion inlet tubing port through its matching slot on the Middle Section i e roughly 7 00 Place the left side narrow slot and wide slot of the Middle Section on top of the matching left side clamp and resister respectively i e roughly 9 00 of the Bottom Section The Middle Section of the DH 35i should now be able to mount directly on top of the Bottom Section without any further restrictions Lift both pieces of flexible tubing from the RC 37 insert to the top of their respective slots in the Middle Section of the DH 35i The flexi
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