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48

4.4 Using the “Tag” Function

4.4.1 To verify counting results after performing the counting function, press “Images” and “Tag”

buttons as shown below.

<Tagged Cell Image>

Green circle – live cell Red circle – dead cell

Press

Images button

Press

Tag button

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Summary of Contents for Luna-FL

Page 1: ...1 Luna FL Automated Cell Counter User Manual Catalog No L20001 Document No L24001 Revision Date July 15 2013 Version 1 0 0 ...

Page 2: ...duct Description 13 Chapter 2 Setting up 16 2 1 Installation 16 2 2 Start Up Screen 17 2 3 Settings 19 2 4 Setting the Date and Time 20 2 5 Save Options 20 2 6 Counting Options 21 2 7 Result Printer Calculator Options 23 Chapter 3 Bright Field Counting Cells 24 3 1 Sample Preparation 24 3 2 Loading Samples into Slides 24 3 3 Counting Cells 25 3 4 Using the Tag Function 29 3 5 Cell Size and Number ...

Page 3: ...ts Chapter 5 Reviewing Saved Count Results 56 58 59 5 1 Importing Previous Counts 59 5 2 Reviewing Previous results 60 5 3 Previous Count button 61 Chapter 6 Setting Up the Protocol for Counting 63 6 1 Adjusting Parameters for Bright Field Counting 63 6 2 Adjusting Parameters for Fluorescence Counting 6 3 Managing Protocols Chapter 7 Maintenance and Troubleshooting 65 66 67 7 1 Cleaning 67 7 2 Cha...

Page 4: ...r connecting both ends of its power cord to the wall outlet as well as the instrument Always turn off the instrument before disconnecting the power cord and or moving the instrument 6 Ensure that the power cord is firmly plugged into the power inlet the wall outlet and AC adapter 7 When the instrument is operating for a long time its temperature can become too high Please be careful that the instr...

Page 5: ...rdinances of your region for proper disposal provisions The CE mark indicates that this instrument conforms to all applicable European Community provisions for which this marking is required Users must be aware of and follow the conditions described in this manual for operating the instrument The protection provided by the instrument may be impaired if the instrument is used in a manner not specif...

Page 6: ... surface Make sure that no damage or contamination occurs on the optical surfaces of the slide 4 After mixing the cell sample with the supplied reagents perform cell counting within 1 3 minutes for accurate cell viability measurements Optimally count your sample at least twice duplicate readings to obtain better results 5 Since the Luna FL is calibrated before shipping users generally do not need ...

Page 7: ...nditions Operating Power 100 240 VAC 1 5A Frequency 50 60 Hz Electrical Input 12 VDC 3 5A Installation Site Indoor use only Operating Temperature 10 35 Maximum Relative Humidity 20 80 Altitude 2 000 m Pollution Degree 2 ...

Page 8: ... PI Acridine Orange and Propidium Iodide from the cell viability kit in case that there is not enough cell sample 9 l of the cell sample with 1 l of the AO PI solution can be used to load the total 10 l in the counting chamber Second load 10 12 l of the mixed cell suspension into the Luna FL cell counting slide Third insert the slide into the slide port of the instrument and adjust the focus knob ...

Page 9: ...as dual fluorescence optical components to provide advanced cell counting functionalities The cell image acquired from each channel bright green and red can be merged directly on the screen The brightness of each color can be adjusted independently for accurate monitoring Small footprint Due to its minimal size 22 cm x 21 x 9 cm weight 1 8 kg it can be used on a clean bench or stored in a biosafet...

Page 10: ...asily be calculated using the built in calculator Set up maintenance Just plug in and it is ready for use with virtually no maintenance time or costs Counting image transfer The counting image can be downloaded onto a USB drive in TIF Tag Image File format for later use or review Individual protocol saving Each user can set or adjust counting parameters and save the counting protocol for later use...

Page 11: ...f 50 slides AO PI cell viability kit 2 X 0 5 ml Trypan blue stain 0 4 2 x 1 ml Luna FL calibration beads 0 5 X 1 ml Luna USB drive 4GB 1 unit When you receive the product package please check that all the components listed above are included and that no damage has occurred during transit The warranty does not cover damage that may occur during the shipping and handling process Any damage claims mu...

Page 12: ...Diameter Range 1 90 optimally 5 60 m Cell Circularity Range 30 100 Cell Viability Range 0 100 Image Resolution CMOS camera 5 MP Image Type TIF Reporting PDF report CSV file LCD Display 7 inches 800 x 480 pixels Processing Time Approx 7 seconds for bright field approx 30 seconds for dual fluorescence may depend on cell type and concentration 1 3 2 Luna FL cell counter counting slide specifications ...

Page 13: ...nctions and displays results Front View 1 4 2 Rear view of the Luna FL automated cell counter The rear view of the Luna FL automated cell counter shows a power button to turn on or off the instrument and a power inlet Connect the instrument to an electrical outlet with the power cord and plug provided in the product package Be sure to check the electrical outlet configuration in your country Rear ...

Page 14: ...cells with dark centers The slide port is used to insert the counting chamber slide loaded with the sample into the instrument Right Side View 1 4 4 Left side view of the Luna automated cell counter On the left side of the cell counter there is a USB drive port into which a USB drive is inserted for transferring and saving data One USB drive is supplied in the product package Any standard USB driv...

Page 15: ... the same sample as duplicates or for two different samples The depth of the counting chamber is 100 m The counted cell volume is about 0 5 l almost the same as counting five 1 mm x 1 mm squares in a standard hemocytometer Note 0 4 trypan blue solution is blue and the AO PI solution is pink or orange Sample loading chamber marked as A and B Sample loading port ...

Page 16: ...omated cell counter on a flat stable surface 2 1 3 Insert one end of the power cord into the instrument and plug the other end of the power cord into an electrical outlet after checking the outlet configuration in your local area 2 1 4 Turn on the instrument using the power button located on the back of the cell counter 2 1 5 The Start Up screen will display on the touch screen as shown below in 2...

Page 17: ... turned on the Start Up screen will display two options one for the Bright Field Cell Counting and the other for Dual Fluorescence Cell Counting Users can choose one of them Start Up Screen of the Luna FL Automated Cell Counter 2 2 2 Bright Field Counting Screen ...

Page 18: ...18 2 2 3 Fluorescence Counting Screen 2 2 3 1 Green Fluorescence Counting Screen 2 2 3 2 Red Fluorescence Counting Screen ...

Page 19: ...gs button located on the upper right corner of the counting screen Then the following Settings screen appears From here users can change or adjust various preferences or options Settings 2 3 3 The Settings menu allows you to perform the following Background calibration of the instrument described in Section 7 3 Calibrating the Counter Firmware update for the installation of new versions of the fir...

Page 20: ... Save Options button As a default only the Analyzed Image button is activated On while the Raw Image and Report button are inactivated Off Save Options 2 5 2 In the Default Image Save menu users can choose their options to save the raw image PDF report or both Each button can be switched on and off Saved Items Description Analyzed image The image contains the counting results and the tagging of li...

Page 21: ...e In case users want to have another default name the Auto header can be customized by editing the default value of Logos Biosystems 2 6 Counting Options 2 6 1 The Counting Options applies only to the bright field cell counting Before selecting an option please make sure that the Bright Field Cell Counting is selected from the options on the Home Screen 2 6 2 In the Counting Options users can choo...

Page 22: ...the dilution factor must be set to value 1 Dilution Factor Check Custom staining In this option cell samples can be stained with other dyes or even the culture media for counting Before choosing this option the instrument must be re calibrated with users own stains or culture media For re calibration please refer to Section 7 3 Calibrating the Counter When using this option make sure that the dilu...

Page 23: ... The default value is off When it is turned on the counting result box displays only the total cell number Dilution Calculator The default value is Total Cells on In order to use the live cells in the Dilution Calculator please turn the live cells on Printer When this button is on the print out of the results has the details of the parameters of counting ...

Page 24: ...Counting mode of Luna FLTM is optimized with the use of trypan blue Although the counting Without Trypan Blue option is provided in the protocol menu section 2 6 2 to meet the various needs of users it is NOT recommended to count cells without trypan blue staining Low contrast due to the lack of the trypan blue staining may sometimes cause abnormal results 3 2 Loading Samples into Slides 3 2 1 Hol...

Page 25: ...it must be taken out rotated and inserted again Make sure that the counting slide is not inserted upside down This may lead to sample spilling and could severely damage the counter 3 3 2 Select the Bright Field Cell Counting button on the home screen Home Screen Note After this step the real image of the cell sample will be displayed on the screen The cell image can be navigated by touching the sc...

Page 26: ...lly the Zoom in button is set to a 1x image When pressed once a 2x image appears This 2x image may be the best zoom in status for focusing When pressed again a 4x image is shown Pressing the button again takes the screen back to a 1x image Note The current staining option is displayed on the left side of the screen as marked by the red box in the image above The staining option can be changed in t...

Page 27: ...ge the preview images to a color mode press the Color button once and the preview image will turn to a color image and the Color button will change to blue Regardless of which preview mode is selected grey or color grey images will be used for calculating the cell number displayed in the cell counting result screen ...

Page 28: ...om in button 3 3 5 Within approximately 7 seconds depending on cell concentration the image of the cell sample and data results Total Live and Dead cell concentrations Viability Avg Cell Size Actual number of Total Live and Dead cells will be displayed on the screen Cell Counting Results ...

Page 29: ...o review data to determine the accuracy of the counting on site without depending on a computer 3 4 2 When touching the Tag button the image on the screen will show objects surrounded by green or red circles The green circles indicate live cells and the red circles indicate dead cells 3 4 3 After reviewing the accuracy of the image analysis Tag button can be pressed again to remove the green and r...

Page 30: ...nal buttons in the menu Count Graph Gating 1 Total Live or Dead button 2 Cluster Map button 3 Cell number or ml button The above buttons can be used in combination and the details are described in next pages When the sizes of the cells are out of the range they are displayed in grey color Combination Total and Cell Number Combination of Total and Cell number shows the distribution of cell size vs ...

Page 31: ...ells Combination Dead Cells and Cell Number The combination of Dead and Cell number shows the distribution of cell size vs cell number for only dead cells Note When the Cell Number button is pressed it toggles to the ml button which shows the cell concentration i e the Y axis of the histogram When pressed again it toggles to the Cell Number button ...

Page 32: ...ation for total cells Combination Live Cells and Concentration The combination of Live vs Cell concentration shows the distribution of cell size vs cell concentration for only live cells Combination Dead Cells and Concentration The combination of Dead vs Cell concentration shows the distribution of cell size vs cell concentration for dead cells ...

Page 33: ...e cells Dead cells 3 5 2 Cluster Map button 3 5 2 1 When the Cluster Map button is pressed the percentage distribution of the clustered cells are displayed 1 cell means single cell and 2 cell means 2 clustered cells as a unit When the Cluster Map button is on the Total and Cell Number buttons become inactivated Cluster map 3 5 2 2 Press the Graph Gating button again to go back to the previous scre...

Page 34: ...lculator initially shows the current concentration Put the appropriate numbers into the blanks of the Desired Concentration and Final Volume that you want to obtain Users can choose one of Total Live or Dead cells for calculating their dilution By clicking this button the Current Concentration displays the concentration of the total live or dead cells Before calculating the dilution factor please ...

Page 35: ...35 Users can also calculate their custom dilution 3 6 3 Press the Calculate button and then a dilution instruction will appear in the message box ...

Page 36: ...Live cells are tagged in green and dead cells are tagged in red for further analysis If not checked users can not use the Review function with the saved image So please make sure that this must be checked if users want to review the saved image on the instrument later Raw Image This box must always be checked in order to save the image in TIF format The saved image can be re analyzed on the instru...

Page 37: ... USB drive The saved files can be opened after transferring the images report via USB drive to a PC with the appropriate software Note With the Luna FL software additional graphical representations of the data and results are provided in the PDF report as shown below Two page PDF report generated by Luna FL 1st page Basic Data and Results Cell counting data Protocol used Cell images tagged and zoo...

Page 38: ...cially designed for the Luna FL and must be purchased only from Logos Biosystems or its local distributors Note For more information on the Luna Printer please see the User Manual of the Luna Printer 3 8 2 First connect the Luna Printer to the Luna FL via the supplied USB connector The Luna Printer can be connected at any time Then turn on the Luna Printer by pressing the Power button for 1 2 sec ...

Page 39: ...39 Print Button 3 8 4 The counting results and its protocol will be printed out as below Printed Results Note The Print button prints only the latest counting results ...

Page 40: ...40 Note If the Protocol part of the print is not necessary users can turn off the option in the Results Printer Calculator Option in the Settings menu as shown below Result Printer Calculator Options ...

Page 41: ...ts 4 1 3 Transfer 2 l of the AO PI staining solution to a new 1 5 ml microfuge tube 4 1 4 Add 18 l of the cell sample to the tube and mix by pipetting up and down or flicking the bottom of the tube while holding with the other hand 4 2 Loading Samples into Slides 4 2 1 Holding the edge of the slide load 10 l of the mixed cell sample into the inlet of one chamber of the counting slide For easy and ...

Page 42: ...3 1 Wait for about 1 min in order for the cells to settle down Note Skipping this step will result in poor counting accuracy Optimal waiting time should be determined empirically If cells are still moving during the preview step step 4 3 4 a longer waiting time is required Note In case that the cells do not settle down the captured images from bright field and green red fluorescence do not align e...

Page 43: ...mber Gently insert the counting slide to the end Note After inserting the slide Luna FL only reads the first chamber To read the second chamber it must be taken out rotated and inserted again Note Make sure that the counting slide is not inserted upside down This may lead to sample spilling and could severely damage the counter 4 3 3 Select the Fluorescence Cell Counting button on the home screen ...

Page 44: ...ich needs a bit of pressure in order to get a response 4 3 4 The Preview screen in the bright field mode will appear as shown in the image below Make sure that users have the best focus When necessary adjust the focus with the focus knob on the right side of the instrument Bright Field Mode in Fluorescence Counting Note Make sure that the bright field mode is selected to adjust the focus Adjusting...

Page 45: ...Increase the GF intensity value if the preview image is too dim so that only some cells are visualized Decrease the GF intensity value if the preview image is too bright so that background fluorescence starts to come up The optimal GF intensity value should be determined empirically The factory set value for mammalian cells is 5 Note During the preview step if cells are irradiated for a long perio...

Page 46: ...rease This bleaching phenomenon is accelerated when a higher level of fluorescence is used Always try to use the shortest preview time and the lowest fluorescence level when the GF or RF preview is selected Red Fluorescence Mode in Fluorescence Counting 4 3 7 Press the Count button 4 3 8 Within approximately 30 seconds depending on cell concentration the image of the cell sample and data results T...

Page 47: ...47 Fluorescence Cell Counting Results ...

Page 48: ...Function 4 4 1 To verify counting results after performing the counting function press Images and Tag buttons as shown below Tagged Cell Image Green circle live cell Red circle dead cell Press Images button Press Tag button ...

Page 49: ...mostly cells surrounded by green or red circles The green circles indicate live cells and the red circles indicate dead cells Note In the Fluorescence Cell Counting mode non cellular debris contained a cell culture medium can be easily identified and automatically excluded from cell counting because it does not have any nucleic acids Non cellular debris 4 4 3 After reviewing the accuracy of the im...

Page 50: ...l buttons in the menu Count Graph Gating 1 Total Live or Dead button 2 Cluster Map button 3 Cell number or ml button The above buttons can be used in combination and the details are described in next pages When the sizes of the cells are out of the range they are displayed in grey color Combination Total and Concentration Combination of Total and Cell number shows the distribution of cell size vs ...

Page 51: ...ells Combination Dead Cells and Cell Number The combination of Dead and Cell number shows the distribution of cell size vs cell number for only dead cells Note When the Cell Number button is pressed it toggles to the ml button which shows the cell concentration i e the Y axis of the histogram When pressed again it toggles to the Cell Number button ...

Page 52: ...ntration for total cells Combination Live and Concentration The combination of Live vs Cell concentration shows the distribution of cell size vs cell concentration for only live cells Combination Dead and Concentration The combination of Dead vs Cell concentration shows the distribution of cell size vs cell concentration for dead cells ...

Page 53: ...ls Dead cells 4 5 2 Cluster Map button 4 5 2 1 When the Cluster Map button is pressed the percentage distribution of the clustered cells are displayed 1 cell means single cell and 2 cell means 2 clustered cells as a unit When the Cluster Map button is on the Total and Cell Number buttons become inactivated Cluster Map 4 5 2 2 Press the Graph Gating button again to go back to the previous screen ...

Page 54: ...alculator initially shows the current concentration Put the appropriate numbers into the blanks of the Desired Concentration and Final Volume that you want to obtain Users can choose one of Total Live or Dead cells for calculating their dilution By clicking this button the Current Concentration displays the concentration of the total live or dead cells Before calculating the dilution factor please...

Page 55: ...55 Users can also calculate their custom dilution 4 6 3 Press the Calculate button and then a dilution instruction will appear in the message box ...

Page 56: ...window contains three check boxes as follows Analyzed Image Check this box to save an analyzed image Live cells are tagged in green and dead cells in red for further analysis Raw Image This box must always be checked in order to save the image in TIF format The saved image can be re analyzed on the instrument later if necessary Report Check this box to generate a printed report as a PDF The report...

Page 57: ...into the USB drive Now the saved files can be opened after transferring them via USB drive to a PC with the appropriate software Note With the Luna FL software additional graphical representations of the data and results are provided in the PDF report as shown below Two page PDF report generated by Luna FL 1st page Basic Data and Results Cell counting data Protocol used Cell images tagged and zoom...

Page 58: ...58 4 8 Printing the Counting Results Please see Section 3 8 Page 38 to print the counting results ...

Page 59: ...1 Importing Previous Counts 5 1 1 Insert the USB drive containing the previous results into the USB port of the instrument 5 1 2 Press the Review menu located at the top of the screen A list of files on the USB drive is displayed on the left side of the screen as shown below Review Screen Note If there are several saved files in the USB drive the scroll bar can be used to navigate the file list ...

Page 60: ...ayed onto the instrument screen Reviewing the previous result 5 2 2 The previous image can be viewed at 1X 2X or 4X by pressing the Zoom in button and navigated by touching and dragging the image window 5 2 3 Press the X close button to review other previous results Or press the Count menu located at the top of the screen and the instrument will be ready to count again ...

Page 61: ...data can be exported as a CSV file to the USB drive for further analysis 5 3 1 Pressing the Previous Count button on the left side of the Review menu will bring up a new window The Previous Count window contains up to 1 000 previous counting results that have been saved in the memory of Luna FL Previous Count Button Previous Count Window ...

Page 62: ...s Count there are 3 buttons 1 Export to USB CSV Users can save the data of all previous counts into a USB memory drive which can be handled in PC software 2 Erase all All data can be removed from the memory 3 Close Close this window ...

Page 63: ... create a customized protocol select New protocol first and edit parameters then save as another name by pressing the Save as button Because the protocol named DEFAULT cannot be edited it must be saved as another name before parameters can be adjusted Note Each counting mode has its own counting parameter set 6 1 Adjusting Parameters for Bright Field Counting When counting is performed the DEFAULT...

Page 64: ...ue solution 1 1 ratio before counting Noise Reduction 0 10 5 This refers to the decrease of the background for counting Higher noise reduction will not detect faint signals and weakly stained objects Lower noise reduction means increasing the sensitivity of the objects and detecting faint signals Adjust the noise reduction to the appropriate level It is also useful when cells are over stained or u...

Page 65: ...u Parameter Range Default Description Dilution Factor 1 10 1 11 The default value for the dilution factor is 1 11 In the protocol of the Fluorescence Cell Counting mode 2 l of the AO PI cell viability kit cat F23001 is added to 18 l of the cell sample The dilution factor in this case is calculated as 1 11 20 18 In case 4 l of the AO PI cell viability kit is added to 16 l of the cell sample the dil...

Page 66: ...old will detect less cells by subtracting the background more stringently Decreased threshold can detect more cells but will increase the number of noise signal Red Fluorescence Threshold 1 10 5 6 3 Managing Protocols To manage protocols use the buttons described below Button Description Load Use this button to load one of the saved protocols to apply for counting or adjusting the parameters Edit ...

Page 67: ...After saving users can load the protocol for future counting Note The Luna FLTM automated cell counter is provided with a factory setting protocol named DEFAULT This DEFAULT protocol cannot be edited or deleted The parameters in the DEFAULT protocol have been tested with many different cell types and can be used for a wide variety of cells without changing parameters ...

Page 68: ...he cell counter with a dry cloth Do not wet the instrument by pouring or spraying water or other liquids directly on the instrument In particular power related parts should never become wet in order to avoid electrical shock or damage 7 1 2 Cleaning the touch screen Gently wipe off the touch screen with a soft cloth lightly moistened with an authorized LCD cleansing detergent Since excessive force...

Page 69: ...ever if the date time changes or slows with unknown causes it may indicate that the battery is weakening or running out To change the battery contact service personnel of the instrument supplier in your country Do not attempt to change the battery in any event Disassembling the instrument voids the product warranty ...

Page 70: ... Counting mode The calibration can be performed at either mode Note Be sure to focus the instrument properly before calibration Use the calibration bead to for the focus adjustment 7 3 2 Go to the Settings menu Press the Calibration button and a message box will appear as shown below Recalibration Note The recalibration consists of 3 steps for bright field calibration custom staining calibration a...

Page 71: ...e counting slide cell counting result will be inaccurate Calibration Step 1 Note Never turn off the Luna FL cell counter during calibration which could lead to a significant technical failure 7 3 4 Calibration Step 2 is necessary for the Custom Staining Calibration From this custom calibration users can use their own stains for cell counting This is related to Section 2 6 Counting Options ...

Page 72: ...10 l of 0 2 concentration Second mix 10 l of 0 2 concentration with another 10 l of PBS to prepare 20 l of 0 1 concentration from which 10 l of 0 1 is loaded into one counting chamber to perform 0 1 Custom Staining Calibration To count cells under 0 1 trypan blue concentration users can mix cells with 0 2 trypan blue solution at a 1 1 ratio Calibration Step 2 2 Note Never turn off the Luna FL cell...

Page 73: ...tion beads into one of the counting chambers and wait for about 1 min in order for cells to completely settle down Insert the counting slide and then press the Start button Make sure to load the original standard solution do not dilute it Calibration Step 3 1 Calibration Step 3 2 Note Never turn off the Luna FL cell counter during calibration which could lead to a significant technical failure ...

Page 74: ...74 7 3 6 When the three calibration steps are done successfully users will see the message box as below Successful Calibration ...

Page 75: ...rmware consisting of 1 file must be saved into the root directory of the USB drive 7 4 3 Insert the USB drive into the USB port on the instrument Then press the Update Firmware button 7 4 4 When the display asks you to update press the Start button The process will take a few minutes for completion 7 4 5 When the update is finished press the Restart button to reboot the instrument Note The firmwar...

Page 76: ...eard Sample loading If the counting slide is over or under loaded with the sample it may affect counting results The optimal amount of sample is 10 12 l of cell suspension Malfunction of optical components Any of the optical components may be damaged Or the objective lens may be dirty due to dust spilled samples or unknown causes Please contact your local supplier Damaged counting surface Make sur...

Page 77: ...en there are too many saved files on the USB drive reading and writing by the counter may slow down Errors during updating and calibrating the instrument Freezing during calibration Generally re calibration takes several minutes However it may take more time depending on the extent of background adjustment If the calibration takes more than 10 minutes reset the system by turning off and on using t...

Page 78: ...78 drive The file should be located in the root directory Third ensure that the USB drive is inserted correctly Last try the update again If the problem continues contact your local supplier ...

Page 79: ...nts 20 boxes B13101 Luna Standard Bead Bright Field Concentration 1 0 X 10e6 ml Size 10 um 2 X 1 0ml F23001 AO PI Cell Viability Kit 500 tests 2 X 0 5ml F23002 AO Cell Viability Kit 500 tests 2 X 0 5ml F23003 PI Cell Viability Kit 500 tests 2 X 0 5ml F23102 Luna FL Calibration Bead 100 tests 1 x 0 5ml F23202 Yeast Viability Kit 1 TBD F23203 Yeast Viability Kit 2 TBD F24004 Luna FL IQ OQ Protocol 1...

Page 80: ...x or email Our regional suppliers are listed on our web page www logosbio com Logos Biosystems Inc Address STE 930 Doosan Venturedigm 126 1 Pyungchon Dong Dongan Gu Anyang City Gyunggi Do Republic of Korea Zip code 431 755 Tel 82 31 478 4185 Fax 82 31 478 4184 E mail sales logosbio com ...

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