Whole cell fluorescence reading Ex485nm/Em530nm with plate reader (SOP ID: 0004)

SOP ID:

0004

Revision nr.:

0.0

Release date:

November 8, 2019

Last release date:

November 8, 2019

Author(s):

Davide De Lucrezia

Approver:

Ada Serena Marletta

1. Scope

This Standard Operating Procedure provides a step-by-step protocol for whole-cell fluorescence reading using a plate reader with Ex485nm/Em530nm. This protocol includes 3 calibration sets (OD600 calibration, Particle standard curve, and Fluorescein standard curve) to ensure that collected data can be exchanged and compared among different laboratories using different equipment at different times. The rationale behind this protocol is to use reference materials and calibration curves to rescale readouts in order to make them directly comparable across different operators/labs/equipment. In particular:

OD600 calibration
LUDOX CL-X (45% colloidal silica suspension) will be used as a single point reference to obtain a conversion factor to transform absorbance data (Abs 600 nm) from your plate reader into a comparable OD600 measurement as it would be obtained in cuvette-based spectrophotometer. Absorbance conversion is required to account for well-to-well volume differences that may occur during plate set up. Any volume difference will affect the path length of the light passing through the sample in the well and hence affect the absorbance measurements. Although most plate readers are equipped with automatic pathlength correction feature, this is performed digitally after data acquisition compromising data reproducibility and comparability among different equipment. For this reason, this SOP requires the pathlength correction to be switched off and be replaced with a single point reference measurement as described in section 7.

Particle standard curve.
A serial dilution of monodisperse silica microspheres with size and optical characteristics similar to cells will be used as a standard curve to measure Abs600 and estimate the number of cells in your well.

Fluorescein standard curve.
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 addition, fluorescence readouts may be affected by process variables such as temperature and humidity. In order to compare the fluorescence output of different devices at different times, it is necessary to rescale readouts measurements to a standard fluorescence curve. This procedure uses the small molecule fluorescein, which has similar excitation and emission properties to GFP, as a surrogate of the GFP protein.
A serial dilution of fluorescein will be used as a standard curve to measure fluorescence with excitation at 485 nm (recommended) and emission 520-530 nm (recommended) with a bandpass width 25-30nm (recommended).

The figure below shows the typical arrangement of a microplate with the 3 standards. Wells A4 to H12 are then used for sample measurements.

2. Background

This Standard Operating Procedure is intended for qualified personnel who was trained according to the ISO-9001 quality framework.

3. Prerequisites

Standard plasticware
fluorescence-compatible 96-well plate

4. Changes since last revision

No changes were made since last version

5. Role and responsibilities

Author (Au): Staff scientist properly trained to draft, review and disseminate standard operating procedures within the framework of ISO-9001

Approver (Ap): PI with the authority to approve, reject and withdraw standard operating procedures

MIAMI Quality Manager (QM): MIAMI authorized Quality Manager

Operator (Op): Staff scientist properly trained to execute the standard operating procedure

Other (Oth): Third-party personnel and/or institution and/or authority, and/or service provider.

6. Monitoring Requirements

The present document can be reviewed at any time required by the author and the MIAMI Quality Manager.

7. Procedure

Step	Action	Responsibility
1	Create OD600 calibration: Add 100 μl LUDOX into wells A1, B1, C1, D1 Add 100 μl of dd H2O into wells E1, F1, G1, H1	Op
2	Create Particle standard curve: Add 100 μl of dd H2O into wells A2, B2, C2, D2, E2, F2, G2 Add 200 μl of dd H2O into wells H2 Resuspend the silica beads by vortex for 30 seconds Immediately transfer 96 μL microspheres into a 1.5 mL eppendorf tube Add 904 μL of ddH2O to the microspheres and vortex Add 100 μL to well A2 and mix by pipetting 3X Transfer 100 μL from well A2 to well B2, mix by pipetting 3X Transfer 100 μL from well B2 to well C2, mix by pipetting 3X Transfer 100 μL from well C2 to well D2, mix by pipetting 3X Transfer 100 μL from well D2 to well E2, mix by pipetting 3X Transfer 100 μL from well E2 to well F2, mix by pipetting 3X Transfer 100 μL from well F2 to well G2, mix by pipetting 3X Important note: DO NOT continue serial dilution into well H2	Op
3	Create Fluorescein standard curve: Add 100 μl of 1xPBS into wells A3, B3, C3, D3, E3, F3, G3 Add 200 μl of 1xPBS into wells H3 Prepare 1000x fluorescein stock solution (10 mM) by resuspending fluorescein in 1mL of 1xPBS Transfer 100 μL of 1000x fluorescein stock solution to 900 μL 1xPBS to make a 100x fluorescein solution (1 mM) Transfer 100 μL of 100x fluorescein stock solution to 900 μL 1xPBS to make a 10x fluorescein solution (100 μM) Transfer 100 μL of 10x fluorescein stock solution to 900 μL 1xPBS to make a 1x fluorescein solution (10 μM) Add 100 μL of 1x fluorescein solution to well A3 and mix by pipetting 3X Transfer 100 μL from well A3 to well B3, mix by pipetting 3X Transfer 100 μL from well B3 to well C3, mix by pipetting 3X Transfer 100 μL from well C3 to well D3, mix by pipetting 3X Transfer 100 μL from well D3 to well E3, mix by pipetting 3X Transfer 100 μL from well E3 to well F3, mix by pipetting 3X Transfer 100 μL from well F3 to well G3, mix by pipetting 3X Important note: DO NOT continue serial dilution into well H3	Op
4	Arrange Cell measurement: For each time point or sample, arrange a triplicate of 200 μL each using wells A4 to H12. The following figure shows samples arrangement for 3 different constructs sampled at 8 different time points	Op
5	Microplate reader set up: Absorbance settings: 600nm Fluorescence settings: excitation at 485 nm (recommended) and emission 520-530 nm (recommended) with a bandpass width 25-30nm (recommended). Temperature settings: 25°C Shaking: 5 sec before each reading (to resuspend particles and cells) Automatic pathlength correction feature: switched off
6	Save raw data as .txt, .csv and proceed to data analysis.	Op

8. Record Management

When completed and approved, the SOP will be published on MIAMI website

9. References

9.1 Standards

9.2 Other SOPs

Sampling procedure for yeast culture (SOP ID: 0012)
Yeast growth protocol (SOP ID: 0010)

9.3 Reagents

LUDOX CL-X (45% colloidal silica suspension) – Sigma Aldrich – 420891

Monodisperse silica microspheres

Fluorescein sodium salt – Sigma Aldrich – 46960

9.4 Sequences

The execution of this SOP does not reference to any reference sequence.

9.5 Supplementary Documents

This SOP is an adaptation of IGEM Plate Reader and CFU Protocol

10. Definitions

Measurements units are defined according to International System of Units
Metric prefixes are defined according to Metric (SI) Prefixes
Whenever possible, biological construct are defined according to the SBOL standard
Whenever possible, biological experiments are defined according to the metrology standards

11. Disclaimers

This Standard Operating Procedure (SOP) was developed based on a compilation of best available information, knowledge, experience, and general industry practices to provide guidance to MIAMI staff in performing the activities defined herein, in a consistent and standardized manner. This document does not contain regulatory or statutory requirements unless specified. The MIAMI consortium has made every attempt to present the information in a clear and concise manner for a variety of users. However, the MIAMI consortium is not responsible for the misuse or misinterpretation of the information presented herein. Under no circumstances shall The MIAMI consortium be liable for any actions taken or omissions made by non-MIAMI users of this document. In general, this document should be used as a reference. Differences may exist between the procedures referenced in this document and what is appropriate under project-specific conditions. This document does not represent an endorsement of practitioners or products mentioned in the document.

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