TITLE: Calibration Protocol - Plate Reader Fluorescence Calibration
AUTHORS: Geoff Baldwin, Traci Haddock-Angelli, Jacob Beal, Ari Dwijayanti, Marko Storch, Natalie Farny, Cheryl Telmer, Alejandro Vignoni, Richard Tennant, Paul Rutten

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">Plate readers report fluorescence values in arbitrary units that vary widely from instrument to instrument. Therefore absolute fluorescence values cannot be directly compared from one instrument to another. In order to compare fluorescence output of test devices between teams, it is necessary for each team to create a standard fluorescence curve. Although distribution of a known concentration of GFP protein would be an ideal way to standardize the amount of GFP fluorescence in our ​E. coli​ cells, the stability of the protein and the high cost of its purification are problematic. We therefore use the small molecule fluorescein, which has similar excitation and emission properties to GFP, but is cost-effective and easy to prepare. (The version of GFP used in the devices, GFP mut3b, has an excitation maximum at 501 nm and an emission maximum at 511 nm; fluorescein has an excitation maximum at 494 nm and an emission maximum at 525nm).</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">You will prepare a dilution series of fluorescein in four replicates and measure the fluorescence in a 96 well plate in your plate reader. By measuring these in your plate reader, you will generate a standard curve of fluorescence for fluorescein concentration. You will be able to use this to convert your cell based readings to an equivalent fluorescein concentration. Before beginning this protocol, ensure that you are familiar with the GFP settings and measurement modes of your instrument. You will need to know what filters your instrument has for measuring GFP, including information about the bandpass width (530 nm / 30 nm bandpass, 25-30 nm width is recommended), excitation (485 nm is recommended) and emission (520-530 nm is recommended) of this filter.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Note: </span><span>The iGEM Abs600 (OD) calibration protocol with microspheres calibration method is a pre-requisite for carrying out this protocol. You will need data from that calibration to analyse the results of this protocol.</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">RFP users</span><span>: This protocol now includes instructions for the use of dyes compatible with some red fluorescent proteins (RFPs). Please note the Measurement committee is still investigating these so this part of the protocol should be considered a work in progress.</span></div></div></div>

[STEPS]
?. [Prepare the fluorescein stock solution]
Spin down fluorescein kit tube to make sure pellet is at the bottom of tube
?. [Prepare the fluorescein stock solution]
Prepare the stock solution of your reference dye. See sub-step 2.1 for Fluorescein and 2.2 for dyes compatible with red fluorescent proteins (note recommendations for these are a work in progress).
?. [Prepare the fluorescein stock solution]
Dilute the 10X reference stock solution with 1X PBS to make a 1X reference working solution with a concentration of 10 μM. E.g. dilute 100 μL of 10X fluorescein stock into 900 μL 1X PBS.
If you are using Rhodamine B, dilute your reference solution to 50 μM instead of 10 μM then continue this protocol normally.
?. [Prepare the serial dilutions of fluorescein]
Accurate pipetting is essential. Serial dilutions will be performed across columns 1-11. Column 12 must contain PBS buffer only. Initially you will setup the plate with the reference working solution in column 1 and an equal volume of 1X PBS in columns 2 to 12. You will perform a serial dilution by consecutively transferring 100 μL from column to column with good mixing.
?. [Prepare the serial dilutions of fluorescein]
Add 100 μL of 1X PBS​ into wells A2, B2, C2, D2....A12, B12, C12, D12
?. [Prepare the serial dilutions of fluorescein]
Add 200 μlL ​of fluorescein 1X stock​ solution into A1, B1, C1, D1
?. [Prepare the serial dilutions of fluorescein]
Transfer 100 μL of fluorescein stock solution from A1 into A2
?. [Prepare the serial dilutions of fluorescein]
Mix A2 by pipetting up and down 3x and transfer 100 μL into A3
?. [Prepare the serial dilutions of fluorescein]
Mix A3 by pipetting up and down 3x and transfer 100 μL into A4
?. [Prepare the serial dilutions of fluorescein]
Mix A4 by pipetting up and down 3x and transfer 100 μL into A5
?. [Prepare the serial dilutions of fluorescein]
Mix A5 by pipetting up and down 3x and transfer 100 μL into A6
?. [Prepare the serial dilutions of fluorescein]
Mix A6 by pipetting up and down 3x and transfer 100 μL into A7
?. [Prepare the serial dilutions of fluorescein]
Mix A7 by pipetting up and down 3x and transfer 100 μL into A8
?. [Prepare the serial dilutions of fluorescein]
Mix A8 by pipetting up and down 3x and transfer 100 μL into A9
?. [Prepare the serial dilutions of fluorescein]
Mix A9 by pipetting up and down 3x and transfer 100 μL into A10
?. [Prepare the serial dilutions of fluorescein]
Mix A10 by pipetting up and down 3x and transfer 100 μL into A11
?. [Prepare the serial dilutions of fluorescein]
Mix A11 by pipetting up and down 3x and transfer 100 μLl into ​liquid waste
Take care not to continue serial dilution into column 12
?. [Prepare the serial dilutions of fluorescein]
Repeat dilution series for rows B, C, D
?. [Measure fluorescence]
Measure the fluorescence of all samples in your plate reader . Ensure that any automatic gain setting is off (if your instrument has one).If you will be using your data in conjuction with measurements from the Plate Reader Abs600 (OD) Calibration protocol, make sure you use the same instrument settings for both protocols.[RFP calibration, work in progress]: Ignore this if you are using fluorescein. If you are not using fluorescein, remember to adjust your plate reader excitation and emissions settings as appropriate for your red fluorescent protein and the alternative dye you will be using. Please also be aware you may need to change the wavelength at which you are taking your OD reading. If your fluorescent protein is absorbing at 600 nm, this will interfere with your Abs600 reading. We recommend measuring your OD at 660 nm instead, if this is beyond the excitation spectrum of your protein. You can check the excitation of many commonly use fluorescent proteins at fpbase.org. Please note OD readings at 600 and 660 nm cannot be directly compared.
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?. [Measure fluorescence]
Record the data in your notebook. Also record the gain setting that you used in your instrument, if available. Please note your standard curve should still work well even if a few of your measurements are saturing the instrument
?. [Measure fluorescence]
Import data into this Excel sheet provided (​fluorescein standard curve tab​):
?. [Congratulations!]
You have now completed this calibration protocol
?. [Prepare the fluorescein stock solution]
Make a 10X fluorescein stock solution (100 μM) by resuspending fluorescein powder in 1mL of 1X PBS. If you are performing an RFP calibration, skip this and to go 2.2 below instead.
It is important that the fluorescein is properly dissolved. To check this, after the resuspension you should pipette up and down and examine the solution in the pipette tip – if any particulates are visible in the pipette tip continue to mix the solution until they disappear.
?. [Prepare the fluorescein stock solution]
[Work in progress]: Skip this step if you are using fluorescein and continue to step 3. Two dyes are in testing by the Measurement committee for RFP calibrations: Texas Red and Rhodamine B.If you are using Texas Red, we recommend you follow the same protocol as fluorescein; make a 100 μM stock solution in PBS and then continue from step 4 below. If you are using Rhodamine B, we recommend you follow this protocol from step 4, but instead of using a 10 μM starting concentration, start with a 50 μM concentration of Rhodamine B and then follow the protocol normally.