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2.2.4 GFP Transfection Assay

2.2.4.1 GFP Transfection Assay

– Bright Field Screen

2.2.4.2 GFP Transfection Assay

– Green Fluorescence Screen

Summary of Contents for LUNA-FL

Page 1: ...1 www logosbio com LUNA FL Automated Fluorescence Cell Counter User Manual Catalog L20001 Document LBSM MD ML LUF 001 Version VL1712 01...

Page 2: ...1 4 Product Description 14 Chapter 2 Setting up 17 2 1 Installation 17 2 2 Start Up Screen 18 2 3 Settings 22 2 4 Setting the Date and Time 23 2 5 Save Options 23 2 6 Bright Field Counting Options 25...

Page 3: ...n 74 5 6 On off of the Bright and Green Channel 77 5 7 Saving the Image and Generating a Report of the Current Count 78 5 8 Printing the Counting Results 80 Chapter 6 Yeast Cell Counting 81 6 1 Sample...

Page 4: ...er 9 Maintenance and Troubleshooting 106 108 9 1 Cleaning 108 9 2 Changing the Battery 109 9 3 Calibrating the Counter 110 9 4 Updating Firmware 9 5 Troubleshooting Chapter 10 Ordering Information 115...

Page 5: ...et 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 wal...

Page 6: ...waste ordinances 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 U...

Page 7: ...the optical 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 min...

Page 8: ...ental Conditions 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...

Page 9: ...Propidium Iodide Stain in case that there is not enough cell sample 9 l of the cell sample with 1 l of the PhotonSlide or LUNA Reusable Slide can be used to load the total 10 l in the counting chambe...

Page 10: ...d 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 accur...

Page 11: ...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 us...

Page 12: ...x 1 mL USB Drive 16 GB 1 unit Note The PhotonSlide is a counting slide developed by Logos Biosystems that provides much better fluorescence signal with ultra low autofluorescencence Users of the LUNA...

Page 13: ...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 Proce...

Page 14: ...r all functions and displays results Front View 1 4 2 Rear view of the LUNA FL Automated Fluorescence Cell Counter The rear view of the LUNA FL Automated Fluorescence Cell Counter shows a power button...

Page 15: ...ight centers and dead cells with dark centers The slide port is used to insert the slide loaded with the sample into the instrument Right Side View 1 4 4 Left side view of the LUNA FL Automated Fluore...

Page 16: ...r two different samples for example the GFP transfection assay The depth of the counting chamber is 100 m The counted cell volume is about 0 5 l almost the same as five 1 mm x 1 mm squares in a standa...

Page 17: ...tomated Fluorescence 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...

Page 18: ...Home Screen When the LUNA FL is turned on the Start Up screen will display 4 options as shown below Users can choose one of them to perform Start Up Screen of the LUNA FL Automated Fluorescence Cell C...

Page 19: ...osbio com 2 2 3 Fluorescence Cell Counting Screen 2 2 3 1 Fluorescence Counting Bright Field Screen 2 2 3 2 Fluorescence Counting Green Fluorescence Screen 2 2 3 3 Fluorescence Counting Red Fluorescen...

Page 20: ...20 www logosbio com 2 2 4 GFP Transfection Assay 2 2 4 1 GFP Transfection Assay Bright Field Screen 2 2 4 2 GFP Transfection Assay Green Fluorescence Screen...

Page 21: ...www logosbio com 2 2 5 Yeast Cell Counting Screen 2 2 5 1 Yeast Cell Counting Bright Field Screen 2 2 5 2 Yeast Cell Counting Green Fluorescence Screen 2 2 5 3 Yeast Cell Counting Red Fluorescence Scr...

Page 22: ...reen Then the following Settings screen appears From here users can change or adjust various preferences or options Settings Note Please make sure that the software version is 3 0 or higher If necessa...

Page 23: ...nly 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...

Page 24: ...o header which is red lined in the above Save Options image is the newly created folder name or file name that all the images start with during the saving process After saving users can see a folder o...

Page 25: ...l viability data Without Trypan Blue When samples do not contain Trypan Blue dye turn on this button and follow the directions in the message boxes These instructions set the Dilution Factor For sampl...

Page 26: ...h Erythrosin B Stain or 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 ple...

Page 27: ...justed automatically even though background of sample is a little brighter or darker Note The LUNA FL basically analyze results based on image so the instrument can provide an accurate counting result...

Page 28: ...ays only the total cell number not showing live dead cells Dilution Calculator Current Concentration The default value is Total Cells on In order to use the live cells in the Dilution Calculator pleas...

Page 29: ...ion is provided in the protocol menu section 2 6 2 to meet mostly the total cell counting for some special users it is NOT recommended to count cells without Trypan Blue staining Low contrast due to t...

Page 30: ...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...

Page 31: ...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...

Page 32: ...he preview image 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 bright blue Regardless of which preview mode is select...

Page 33: ...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 numb...

Page 34: ...s the user to review the 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...

Page 35: ...al buttons in the menu Count Graph Gating 1 Total Live or Dead button 2 Cluster Map button 3 Cell number or Cell concentration button The above buttons can be used in combination and the details are d...

Page 36: ...bination 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 toggle...

Page 37: ...oncentration 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 Combin...

Page 38: ...ells Live 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...

Page 39: ...he dilution calculator 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...

Page 40: ...40 www logosbio com 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 41: ...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 o...

Page 42: ...eport into the 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 repr...

Page 43: ...e specially 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 Pri...

Page 44: ...44 www logosbio com 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 45: ...ogosbio com 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...

Page 46: ...results 4 1 3 Transfer 2 l of the Acridine Orange Propidium Iodide Stain 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 b...

Page 47: ...very important Note Skipping this step may lead to poor counting accuracy because moving cells can generate poorly aligned cell images from the bright field green and red channel respectively Optimal...

Page 48: ...erted chamber 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...

Page 49: ...is a resistive touch screen which 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...

Page 50: ...ton once to preview the green fluorescence GF image 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 previe...

Page 51: ...celerated 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 Fluor...

Page 52: ...52 www logosbio com Fluorescence Cell Counting Results...

Page 53: ...he 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...

Page 54: ...show objects 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 deb...

Page 55: ...in the menu Count Graph Gating 1 Total Live or Dead button 2 Cluster Map button 3 Cell number or Cell concentration button The above buttons can be used in combination and the details are described in...

Page 56: ...bination 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 toggle...

Page 57: ...l concentration 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 Combinati...

Page 58: ...ive cells 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...

Page 59: ...The dilution calculator 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...

Page 60: ...60 www logosbio com 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 61: ...d 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...

Page 62: ...or report 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 represent...

Page 63: ...63 www logosbio com 4 8 Printing the Counting Results Please see Section 3 8 to print the counting results...

Page 64: ...x 106 cells mL If the initial concentration is too high add some cell culture medium to dilute If the initial concentration is too low centrifuge the cell suspension and resuspend the cells in a smal...

Page 65: ...of the negative control on one side and the GFP transfected sample on the other side of the counting slide For easy and accurate loading tilt the pipette around 45 60 degrees as shown below Sample Lo...

Page 66: ...red images from bright field and green fluorescence do not align exactly as shown below Unsettled Cells 5 3 1 2 Insert the slide containing the GFP transfected cells into the slide port of the instrum...

Page 67: ...e that the touch screen of the LUNA FL Automated Fluorescence Cell Counter is a resistive touch screen type which needs a bit of pressure in order to get a response 5 3 1 4 The Preview screen in the b...

Page 68: ...ving 5 3 1 5 Press the BF GF selection button once to preview the green fluorescence GF image Increase the Green exposure level if the preview image is too dim so that only some cells are visualized D...

Page 69: ...usted 5 3 2 Setting the negative control 5 3 2 1 Remove the slide containing the transfected sample and insert a slide containing the negative control 5 3 2 2 Press the Set control button Note A pop u...

Page 70: ...epending on cell concentration the image of the cell sample and data results Total concentration GFP concentration GFP percentage Total cell number GFP number Avg size Dil Factor will be displayed on...

Page 71: ...esult Screen 5 3 3 3 If you have multiple GFP transfected samples and want to measure the efficiency of the next sample insert the new slide and press the Next Count button 5 3 3 4 Press the Count Sam...

Page 72: ...Note This Tag function is one of the distinct tools of the LUNA FL Automated Fluorescence Cell Counter because it helps users review and determine the accuracy of measuring GFP transfection efficienc...

Page 73: ...www logosbio com Tagged Cell Image 5 4 3 After reviewing the accuracy of the image analysis Tag button can be pressed again to remove the green circles Green circle GFP transfected cell Press Tag butt...

Page 74: ...ear with more details on the GFP intensity and number of the cell sample as shown below GFP transfected cells are displayed as a green line and negative control cells are displayed as a grey line Grap...

Page 75: ...he threshold drawn automatically is not satisfactory users can re assign the threshold as shown in the next figure to meet their specific needs User defined GFP Intensity Threshold 1 GFP Intensity Thr...

Page 76: ...hould be pressed Please note that the percentage values of GFP and GFP cells changed from the previous figures GFP intensity of the control cells When the Control Sample button is pressed once the GFP...

Page 77: ...osbio com 5 6 On off of the Bright and Green Channel 5 6 1 Users can turn on or off of the bright and green channels each by clicking the On Off button Bright Field on and Green on Bright Field off an...

Page 78: ...d was lowered 5 7 Saving the Image and Generating a Report of the Current Count 5 7 1 To save data and or generate a report insert a USB drive into the USB port on the left side of the instrument 5 7...

Page 79: ...a PDF The report contains all the information of the cell counting session This report can be read and printed by a personal computer PC Please un check if a report is not necessary Note Remember to...

Page 80: ...re provided in the PDF report as shown below Two page PDF report generated by LUNA FL 1st page Data and Results Cell counting GFP data Protocol used Cell images tagged and zoomed 2nd page Fluorescence...

Page 81: ...0 l 20 Shelf life 16 months from manufacture date if stored appropriately FDA is dissolved in DMSO User precaution is required Fluorescence signal enhancer 1 can be toxic if exposed to human skin Glov...

Page 82: ...nzymes Esterase of cells Whereas the amount nucleic acid does not vary greatly between different yeast strains the activity of metabolic enzymes can vary in much higher degree between different yeast...

Page 83: ...nlet of one chamber of the counting slide For easy and accurate loading tilt the pipette around 45 60 degrees as shown below Sample Loading 6 2 2 The remaining 10 l of the sample can be used for anoth...

Page 84: ...d chamber is inserted first into the slide port The instrument analyzes only the first inserted chamber Gently insert the counting slide to the end Slide Inserting Note After inserting the slide the L...

Page 85: ...step may lead to poor counting accuracy because moving cells can generate poorly aligned cell images from the bright field green and red channel respectively Optimal waiting time should be determined...

Page 86: ...ly determine the parameters 6 3 5 Touch the Count button Yeast Counting Results Note Please refer to the following counting case S Cerevisiae was counted perfectly with PI FDA at exponential growth st...

Page 87: ...ating 3 60 m Green fluorescence threshold 5 Red fluorescence threshold 5 6 3 6 By clicking the Graph Gating button the screen will show the Size Number distribution as shown below Yeast Size Number Di...

Page 88: ...88 www logosbio com Adjusting the Max size Note By clicking the Total Live Dead button users can see only one of the total live or dead cell groups as below Live Cells Only...

Page 89: ...89 www logosbio com Dead Cells Only Note By clicking the Cluster Map button users can see the grouping status of the Cluster Map as below Cluster Map...

Page 90: ...w Result Screen Note This Tag function is one of the distinct tools of the LUNA FL Automated Fluorescence Cell Counter because it helps users review and determine the accuracy on site without dependin...

Page 91: ...ch light channel can be changed by adjusting the arrowhead buttons Turn off of the Bright Field Channel 6 4 3 After reviewing the accuracy of the image analysis Tag button can be pressed again to remo...

Page 92: ...otocol Using the Fluorescence Signal Enhancer 1 Cat F23213 1 Dilute yeast cells up to 1 100 with the LUNA FL dilution buffer Or centrifuge the yeast culture and resuspend in the LUNA FL dilution buffe...

Page 93: ...93 www logosbio com 6 6 Printing the Counting Results Please see Section 3 8 to print the counting results...

Page 94: ...n applies to all 4 counting categories 7 1 Importing Previous Counts 7 1 1 Insert the USB drive containing the previous results into the USB port of the instrument 7 1 2 Press the Review menu located...

Page 95: ...be displayed onto the instrument screen Reviewing the previous result 7 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 i...

Page 96: ...can be exported as a CSV file to the USB drive for further analysis 7 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 co...

Page 97: ...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 First insert a USB drive and then click the Export to...

Page 98: ...ument To 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...

Page 99: ...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 redu...

Page 100: ...d Minimum Cell Size 1 90 3 Use this parameter to set the minimum cell size for inclusion The unit is 1 micrometer Maximum Cell Size 1 90 60 Use this parameter to set the maximum cell size for inclusio...

Page 101: ...in this case is calculated as 1 11 20 18 In case 4 l of the Acridine Orange Propidium Iodide Stain is added to 16 l of the cell sample the dilution factor is 1 25 20 16 When the amount of the Acridin...

Page 102: ...uorescence Threshold 1 10 5 Green and Red Fluorescence Threshold will determine the level of threshold during the image processing Increased threshold will detect less cells by subtracting the backgro...

Page 103: ...le the dilution factor is 1 25 20 16 Likewise the dilution factor changes from 1 11 2 l of the FDA PI cell viability kit used 1 25 4 l 1 42 6 l 1 66 8 l 2 00 10 l 2 50 12 l 3 33 14 l 5 00 16 l to 10 0...

Page 104: ...orescence Threshold 1 10 2 the background more stringently Decreased threshold can detect more cells but will increase the number of noise signal For yeast it is recommended to start with 2 or 3 depen...

Page 105: ...example 10 times diluted samples should have the dilution factor of 20 2 x 10 Dilution factor can be adjusted either by a scale of 1 or a scale of 10 between 2 to 10 and 10 100 respectively Dilution f...

Page 106: ...ion The unit is 1 micrometer Maximum Cell Size 1 90 60 Use this parameter to set the maximum cell size for inclusion The unit is 1 micrometer 8 5 Managing Protocols To manage protocols use the buttons...

Page 107: ...tomated Fluorescence 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 wi...

Page 108: ...ediately dry the 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...

Page 109: ...10 years However 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 the service personnel of the instrumen...

Page 110: ...unting 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 9 3 2 Go to the Settin...

Page 111: ...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 9 3 4 Calibration...

Page 112: ...prepare 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 perfo...

Page 113: ...rs 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 Cal...

Page 114: ...114 www logosbio com Successful Calibration...

Page 115: ...ote The firmware consisting of 1 file must be saved into the root directory of the USB drive 9 4 3 Insert the USB drive into the USB port on the instrument Then press the Update Firmware button 9 4 4...

Page 116: ...f 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 opti...

Page 117: ...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 o...

Page 118: ...118 www logosbio com correctly Last try the update again If the problem continues contact your local supplier...

Page 119: ...Slide Coverslips 10 units Beads B13101 LUNA Standard Beads 2 x 1 mL F23102 LUNA Fluorescence Calibration Beads 1 x 0 5 mL Reagents F23001 Acridine Orange Propidium Iodide Stain 2 x 0 5 mL F23002 Acri...

Page 120: ...he purchaser of infringement of patents owned or controlled by the Company which cover the product based on the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic pro...

Page 121: ...aphies or other kinds are inevitable In addition the Company reserves the right to make any changes necessary without notice as part of ongoing product development If you discover an error in any of o...

Page 122: ...185 Fax 82 31 478 4184 Email sales logosbio com USA 7700 Little River Turnpike STE 207 Annandale VA 22003 USA Tel 1 703 622 4660 Tel 1 703 942 8867 Fax 1 517 266 3925 Email sales logosbio com EUROPE 1...

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