BioTek Flx800 - Siloam Biosciences
Contents
1. ewrererereerereeeee 1 Enter Manually I Read From File 1 Ready User Note There are several options for collecting data from the BioTek Gen5 software Data can be exported directly to excel or appropriate macros can be prepared within the BioTek software For more information on data output options please refer to reader user manual or contact BioTek Technical Support Sensitivity Adjustment When defining reading parameters for Fluorescence analysis setting the PMT sensitivity or gain in some types of fluorescence reader is important for obtaining useful measurements BioTek recommends a setting between 40 120 for Fluorescence assays Siloam s OptiGlow is a strong chemifluorescence substrate which requires low value on sensitivity setting A manual sensitivity gain setting is recommended for reading Optimiser microplates Following is an instruction for determining the sensitivity setting with mixture of HRP and OptiGlow substrate It use BioTek FIx800 as an example but can be applied to other fluorescence microplate reader as well Material 1 Siloam s SAv HRP cat OMR HRP Note Any HRP conjugate with concentration greater than 1 ug mL can be used for this test with following experimental protocol 2 Siloam s OptiGlow substrate 3 One Optimiser Microplate Well A1 will be used for the sensitivity adjustment SILOAM BIOSCIENCES INC Page 9 of 13 DOC ID2. 7 Anew window will again appear this is the experiment window Insert your plate to be read and click on the Read Plate icon with the small green arrow second from last icon in the lower toolbar Ss Gen5 Experiment2 created from Optimiser_QuantaRed prt Fie Plate Protocol Window System Help z both ds gi KIAIA Plate 1 El gt Protocol E Procedure Xp Plate Layout E Data Reduction ee ee rs Runtime Prompts EE Report Builder Ez File Export Builder Power Export Builder B Data Views Protocol Options g Plate 1 a Information fm Sample IDs Calculation Log 2 Audit Trail sA Audit Trail s as A Ready o i E o j EE a j start S9 Gen5 Experiment2 Be Gen5 OC Destrozada Erika Riv nih Microsoft Excel lt Ve Mi K 3 10 PM SILOAM BIOSCIENCES INC Page 8 of 13 DOC ID OPTI 2 MS 0006 A1 www siloambio com 8 One last popup will appear Select Read and instrument will commence reading your plate se Gen5 Experiment2 created from Optimiser_QuantaRed prt File Plate Protocol Window System Help AU ZAHM ODS SGHSeRans FS Plate 1 Protocol Matrix Statistics H Plate 1 D Audit Trail Reading Date Tuesday August 17 2010 14 42 01 Plate ID Barcode Runtime prompts Number of Samples Comments
3. Fluorescence and Endpoint from the drop down menus Select the proper filters Top for optic position and input 45 for sensitivity as shown below Also select the wells of your plate you wish to read by clicking on the Full Plate icon to the left s Gen5 Protocol1 em Help FUSER TES ie D 2 Procedure gt Plate Layout Data Reduction Ca Runtime Prompts Report Builder B File Export Builder E Power Export Builder DS Data Views Protocol Options Dispense T wi a p Delay Kinetic Monitor Well Plate Out In Stop Resume uw 4 oO a fa y Cc a Comment Synchronized Modes Well A Procedure FLx800 Com9 Plate Type 96 WELL PLATE Advanced Options Read Step Step Label I Detection Methods Fluorescence Read Type i Endpoint Filter Sets 1 Excitation 528 20 I Emission i 590 35 1 Optics Position Top Sensitivity 145 ss Filter Switching per ell Ready gp Gen5 Protocoli User Please see Sensitivity Adjustment at Page 9 SILOAM BIOSCIENCES INC www siloambio com Page 5 of 13 DOC ID OPTI 2 MS 0006 A1 5 Your screen should now look like the picture below and then select OK In the upper left under File save your protocol T Gen5 Protocol AB H E I EEEE D g Procedure p gt Plate Layout Data Reduction F Procedure F
4. OPTI 2 MS 0006 A1 www siloambio com Experimental Procedure 1 Ina clean plastic tube add 50 uL of OptiGlow A 50 uL of OptiGlow B 1 uL of OptiGlow C and 1 uL of supplied SAv HRP stock solution mix well and wait for 2 minutes The substrate will be fully developed and stable for hours 2 Load 4 uL of mixture into well A1 of Optimiser microplate and wait until the well is empty do not use pad holder 3 There are two methods to determine the sensitivity with BioTek FIx800 a Try different sensitivity manually so that the signal from well A1 is close to 11 000 b Use auto sensitivity adjustment An example operation for BioTek FIx800 is shown as below e Follow the instruction of Instrument Setup at Page 3 in step 4 select the option tab under the sensitivity instead of setting the sensitivity manually 73 Read Step Step Label lt default gt i Al A12 Detection Method Fluorescence Read Type Endpoint Filter Sets 1 Excitation 528 20 v Emission Optics Position Top Sensitivity e Your screen should now look like the picture below Select Automatic Sensitivity Adjustment and Scale to High Wells Also select A1 as the reference well by clicking on the tab next to Scale Wells Input 11000 The reader will automatic choose the gain sensitivity which makes the reading of A1 close to the given value LTE isG Hepas SS Usecti
5. nm Srinetiates Readers with pre configured optical set select the wavelength setting for Rhodamine or Cy3 Step 2 Selecting the plate type Optimiser microplate fits 96 well SBS standard in all specifications Please use 96 well standard or similar in plate type setting Step 3 Selecting the probe direction Please use top reading for probe direction Step 4 Selecting the sensitivity gain When defining reading parameters for fluorescence analysis setting the PMT sensitivity or gain in some types of fluorescence reader is important for obtaining useful measurements A manual sensitivity gain setting 1 The gai is recommended for reading Optimiser microplates The procedure is as described below In a clean plastic tube add 50 uL of OptiGlow A 50 uL of OptiGlow B 1 uL of OptiGlow C and 1 uL of Siloam s SAv HRP stock solution mix well and wait for 2 minutes The substrate will be fully developed and stable for hours Note Any HRP conjugate with concentration greater than 1 ug mL can be used for this test with following experimental protocol Load 4 uL of mixture into one well of Optimiser microplate and wait until the well is empty do not use pad holder Read that well in reader with various gain setting Select the gain which gives the RFU reading closest to 11 000 Use the same gain setting read one blank well of Optimiser the readout should be less than 50 Save or r
6. Lx800 Com9 Cal Runtime Prompts Bas Add Step n Report Builder Plate Type 36 WELL PLATE v Advanced Options E File Export Builder Read Power Export Builder M Data Views Protocol Options Cuvette Dispense Description Comments Read F 528 20 590 35 Delay Monitor Well in Pca y Q i Set Temperature Plate Out In Stop Resume Comment Synchronized Modes Well Ready User GB Gens Protocol SILOAM BIOSCIENCES INC Page 6 of 13 DOC ID OPTI 2 MS 0006 A1 www siloambio com 6 To run an actual experiment click on File again and select New Experiment from the drop down list A smaller window will appear select your protocol and click OK s Gen5 Experiment3 File Plate Protocol Window System Help ABEBPH ASS HASBads Cf MS New Experiment Select a Protocol Gen5 Protocol Panel Name Type Modified Folder gt Default Protocol Optimiser QuantaRed prt Gen5 Protocol 8 17 2010 2 40 0 C Program Files BioTek Gen6 gt colume one by one prt Gen5 Protocol 5 20 2010 11 26 C Program Files BioTek Gen6 D prot alpha prt Gen5 Protocol 11 30 2009 12 01 C Program Files BioT ek Gen6 D test2 prt Gen5 Protocol 11711 2009 12 51 C Program Files BioT ek Gen6 Ready eles go 3emnS E periment2 He Gen Ex periments SILOAM BIOSCIENCES INC Page 7 of 13 DOC ID OPTI 2 MS 0006 A1 www siloambio com
7. TECHNICAL NOTE TN0002 Optimiser Microplate System ELISA Setup Guide on the BioTek FLx800 Fluorescence Microplate Reader THE NEXT GENERATION OF MICROPLATES Better Immunoassays through Innovative Microfluidics Table of Contents READER SETUP WITH BIOTEK FIX800 READER ssccccccccsssssecceccsseesseeccceesseesseeeccessueusseeeeessseueeeecessseeeassecesssauaeaeeseeeeaaas 3 RECOM MOC ODU S aise rp aatere tenner danas E E T E E A T 3 e UMS SM E EE E E E E EA 3 SAPEVA S E e E E E E E E A 9 GENERALREADER SETUP CUIDANCE i crte cua ronnctoetactssnusenss vote E N E T EE N 12 SILOAM BIOSCIENCES INC Page 2 of 13 DOC ID OPTI 2 MS 0006 A1 www siloambio com READER SETUP WITH BIOTEK FIX800 READER The BioTek FLx800 Fluorescence Microplate Reader has been tested for compatibility with Siloam s Optimiser microplate System in ELISA assay with OptiGlow chemifluorescent substrate Please refer to the Optimiser Technology page on Siloam s website for more details on the principles behind the Optimiser microplate platform For more detailed information and technical support of BioTek instruments or Gen5 software please contact BioTek Instruments at 1 888 451 5171 a BioTek Instruments Part Number Wavelength Excitation 7082247 528 20 nm or similar Emission 7082224 590 35 nm or similar Instrument Setup 1 Turn on the plate reader and open up BioTek Gen5 software on compute
8. ecord this gain setting This defines the max reading RFU max that Optimiser based assays can reach with this reader gain sensitivity setting n setting will be valid for all Optimiser based assays Repeat Step 4 if a changing the reader or b changing the optical unit such as light bulb filters etc SILOAM BIOSCIENCES INC Page 12 of 13 DOC ID OPTI 2 MS 0006 A1 www siloambio com Technical Assistance If you require assistance please contact Siloam Biosciences Inc Technical Support at 513 429 2976 or techsupport siloambio com Additional technical assistance is available under the Technical Support tab on the Siloam Biosciences web site http siloambio com Using Optimiser Immunoassay Microplate Video e Optimiser User s Guide e Reader Settings e Quick Reference Guide e Frequently Asked Questions e Application Notes Two additional videos appear under the Technology tab of the web site e Optimiser Principles of Operation e Running an Assay with Optimiser Better Immunoassays Through Innovative Microfluidics SILOAM BIOSCIENCES INC www siloambio com SILOAM Page 13 of 13 Siloam Biosciences Inc 413 Northland Blvd Cincinnati OH 45240 Phone 513 429 2976 Fax 513 429 2946 Www siloambio com DOC ID OPTI 2 MS 0006 A1
9. g will be valid for all Optimiser based assays Readjust sensitivity only if a changing the reader or b changing the optical unit such as light bulb filters etc SILOAM BIOSCIENCES INC Page 11 of 13 DOC ID OPTI 2 MS 0006 A1 www siloambio com GENERAL READER SETUP GUIDANCE Optimiser based assays are compatible with standard fluorescence plate readers and multi mode plate readers with fluorescence reading capability Below is the general guidance for setting up the readers For further assistance please contact Siloam s technical support Step 1 Selecting the wavelength for excitation and emission light Assays on Optimiser uses OptiGlow substrate which can be detected using the appropriate excitation and emission settings Figure 5 Quantitation does not require filters that precisely match the excitation emission maxima However a non overlapping filter set with a bandpass that includes the excitation emission spectra is required Wavelengths at 530 575 nm for excitation and 585 630 nm for emission can be used for detection Below are examples for different types of A 7 PADI i l o Filter based readers install 528 20 nm or similar filter for Figure 5 Normalized absorption excitation and 590 35 nm or similar filter for emission left and emission right spectraof Monochromator based readers in wavelength setting set OptiGlow chemifluorescent excitation at 528 20 nm and emission at 590 35
10. r 2 Select Protocol in the Create a New Item menu Recycle Bin Mozilla Firefox Experiment QC xpt Experiment2 xpt sdsd xpt N wizard Experiment1 xpt 3 Read a Plate More z System Test a iS Protocol Utorilals ee colume one by one prt eee Absorbance prot alpha prt Fluorescence ee More Luminescence V ah SILOAM BIOSCIENCES INC Page 3 of 13 DOC ID OPTI 2 MS 0006 A1 www siloam 1 3 A blank protocol will open Select Procedure on the left hand side and a new window will open up Select 96 WELL PLATE in the plate type Optimiser plate fits 96 well SBS standard in all specifications Please use the similar 96 well plate type option in other fluorescence readers such as 96 well plate 96 well standard or 96 well SBS standard ry Gen5 Protocol ALLAN HeeeeDs s ae Procedure Piste Layout iB Data Reduction F Procedure FLx800 Com9 CA Runtime Prompts Report Builder E File Export Builder Read By Power Export Builder M Data Views Protocol Options Add Step Dispense Description Comments Delay Monitor Well Shake Set Temperature Plate Out In Stop Resume Comment wn lt pal T fa 2 N a ao a T wy Ready User GB Gens Protocolt SILOAM BIOSCIENCES INC Page 4 of 13 DOC ID OPTI 2 MS 0006 A1 www siloambio com 4 Select the Read tab Select
11. rst fileuset sensitivity from THIS Read Step L N O Scale to High Wells i Scale to Low Wells Scale Wells Measurement Options Delay after plate movement 100 msec Measurements per data point 10 Delay between measurements 1 msec t The RFU max Value of 11 000 is selected for readers with linear operating range up to 100 000 RFU Setting RFU max 11 000 will allow users to a compare results with Siloam s data and allow for effective troubleshooting if needed and b use the wide dynamic range protocols as described in Application Note ANO0O15 Optimiser Assays with Extended Dynamic Range SILOAM BIOSCIENCES INC Page 10 of 13 DOC ID OPTI 2 MS 0006 A1 www siloambio com e And then find the sensitivity value by right clicking the Procedure and selecting AutoSensitivity Results and use the number for setting the sensitivity ss Gen5 OC File Plate Protocol Window System Help aU2anH ae lt AutoSensitivity Results Filter Set ie sensitivity 1 1 45 4 Plate 1 Kena E Protocal Matrix Chatis er Piste L Edit E ke j AutoSensitivity Results Ek Data Ae Mal Runtime Promots 4 Use the same sensitivity setting read one blank well of Optimiser the readout should be less than 50 5 Save or record this sensitivity setting 6 This defines the max reading RFUmax that Optimiser based assays can reach with this reader sensitivity setting The gain settin
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