TITLE: PLATERO - Green Fluorescence Calibration in Plate Readers
AUTHORS: Alejandro   Vignoni, Yadira  Boada

[DESCRIPTION]
One of the most common sources of information in Synthetic Biology is the data coming from plate reader fluorescence measurements. These experiments provide a measure of the light emitted by certain fluorescent molecules, such as the Green Fluorescent Protein (GFP). However, these measurements are generally expressed in arbitrary units, and are affected by the measurement device gain. This limits the range of measurements in a single experiment and hampers the comparison of results among experiments. In this work, we provide a calibration protocol to express fluorescence measures in concentration units of a reference fluorophore. The protocol removes the gain effect of the measurement device on the acquired data. In addition, the fluorescence intensity values are transformed into units of concentration using a Fluorescein calibration model. Both steps are expressed in a single mathematical expression which returns normalized, gain independent, and comparable data, even if the acquisition was done at different device gain levels.  The protocol embeds a Lineararity and Bias Analysis that provides an assessment of the uncertainty of the model estimations, and a Reproducibility and Repeatability analysis that evaluatesthe sources of variability originating from the measurements and the equipment. All the functions used to build the model, exploit it with new data, andperform the uncertainty and variability assessment are available in an open access repository.

[GUIDELINES]
For a full set of calibrations, you should run two protocols: this fluorescence calibration curve with Texas Red, and the Abs600 (OD) calibration with microspheres. 

Before beginning these protocols, please ensure that you are familiar with the measurement modes and settings of your instrument. For all of these calibration measurements, you must use the same plates and volumes that you will use in your cell-based assays. You must also use the same settings (e.g., filters or excitation and emission wavelengths) that you will use in your cell-based assays. If you do not use the same plates, volumes, and settings, the calibration will not be valid.

Make sure to record all information about your instrument to document your experiment. If your instrument has variable temperature settings, the instrument temperature should be set to room temperature (approximately 20-25 C) for all measurements.

[STEPS]
SECTION: Stock Reference Fluorescein solution
1. Start from at least 1 mL  of 10 micromolar (µM)     solution in  . 

SECTION: Prepare the serial dilutions of Fluorescein
4. Accurate pipetting is essential. Serial dilutions will be performed in 5 tubes. There will be a sixth tube that must contain PBS buffer only. Initially, you will setup 5 tubes labeled Dilution 2 to 5 and PBS,  with 2 mL  of PBS.
Then you will perform a serial dilution by consecutively transferring 2 mL from Dilution 1 to Dilution 2 and so on, always with good mixing (pipetting up and down three times or more).

   
  

SECTION: Stock Reference Fluorescein solution
2. [OPTIONAL] If you have access to a spectrophotometer, you can calculate the concentration of your Fluorescein reference stock solution even more accurately using the Beer-Lambert law. 
SECTION: Stock Reference Fluorescein solution
2.1. Measure the solution's absorbance at 492 nm and calculate concentration using an extinction coefficient of 68.029 mM-1 cm-1

SECTION: Prepare the starting dilution of Fluorescein solution
3. Dilute 250 µL of the  stock reference solution into 3750 µL of PBS to obtain 4 mL  of Dilution 1 @  0.625 micromolar (µM) 

SECTION: Stock Reference Fluorescein solution
2.2. 
If the concentration of your stock reference solutions is different than 10 micromolar (µM) then you need to recalculate the dilution of the next step to obtain a Dilution 1 solution of 0.625 micromolar (µM) 

SECTION: Prepare the serial dilutions of Fluorescein
4.2. Add 2 mL of PBS  to all the tubes.
SECTION: Prepare the serial dilutions of Fluorescein
4.1. Label four tubes with the names Dilution 2 to 5, and one tube with the name PBS.
SECTION: Prepare the serial dilutions of Fluorescein
4.3. Transfer 2 mL of Dilution 1 into Dilution 2 tube. Mix thoroughly by pipetting up and down three times or more.

SECTION: Prepare the serial dilutions of Fluorescein
4.4. Transfer 2 mL of Dilution 2 into Dilution 3 tube. Mix thoroughly by pipetting up and down three times or more.
SECTION: Prepare the serial dilutions of Fluorescein
4.5. Transfer 2 mL of Dilution 3 into Dilution 4 tube. Mix thoroughly by pipetting up and down three times or more.
SECTION: Prepare the serial dilutions of Fluorescein
4.6. Transfer 2 mL of Dilution 4 into Dilution 5 tube. Mix thoroughly by pipetting up and down three times or more.
SECTION: Prepare the serial dilutions of Fluorescein
5. Transfer 2 mL of Dilution 5 into the liquid waste.


SECTION: Micro-plate set up
6. Transfer 100 µL of each dilution into the corresponding well using the following plate map:
  

 

 

SECTION: Measure Fluorescence
7. Measure the fluorescence of all samples in your plate reader, repeat measurements using 4 different gain settings, and then repeat everything 8 times.   

 


SECTION: Record data
8. Save your measurements into an excel file with each repetition on a different sheet.