Optimized Protocol for Microalgae DNA Staining with SYTO9/SYBR Green I, Based on Flow Cytometry and RSM Methodology: Experimental Design, Impacts and Validation
Abstract
:1. Introduction
2. Experimental Design
2.1. Materials and Reagents
- Chromochloris zofingiensis SAG 211-14 (SAG culture strains collection, Göttingen, Lower Saxony, Germany).
- Dimethyl sulfoxide (DMSO) ROTIPURAN® ≥ 99.8% (Carl Roth GmbH + Co. KG, Karlsruhe, Baden-Württemberg, Germany, article number 4720.1).
- Bristol’s Modified (BM) medium (see [60] for the detailed composition).
- 0.9% NaCl solution.
- SYBRTM Green I Nucleic Acid Gel Stain (Thermo Fisher Scientific, Waltham, MA, USA; Cat.no.: S7563).
- SYTOTM 9 Green Fluorescent Nucleic Acid Stain (Thermo Fisher Scientific, Waltham, MA, USA; Cat.no.: S34854).
2.2. Equipment
- CyFlow Cube 8, equipped with 488 nm solid laser (SYSMEX GmbH, Norderstedt, Schleswig-Holstein, Germany).
- Eppendorf Thermomixer Comfort (Eppendorf AG, Hamburg, Germany; Cat.no.: 5355).
- GENESYSTM 150 UV-Visible Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA; Cat.no.: 840-300000).
3. Procedure
3.1. Strain Cultivation Conditions
3.2. Preparation of Stock Solutions
- The stock solutions of SYBR Green I (100 X) and Syto 9 (10 µM) were prepared in DMSO in opaque plastic Falcon tubes, from an initial commercial solution of (10,000 X) and (5 mM), respectively. They were transferred to 1.5 mL, opaque Eppendorf tubes and frozen at −20 °C, for storage.
- ⚠ CRITICAL STEP: it is preferable to avoid the use of glass materials during preparation to prevent dye adsorption on the walls.
- ⚠ CRITICAL STEP: both dyes should be protected from light when preparing, using and storing the stock solutions to avoid their photodegradation.
3.3. Sample Preparation
- 2.
- Cell broth from the shake flasks was diluted in a 0.9% NaCl solution, in a plastic Falcon tube, to a cell density of 7.5 ∗ 106–8 ∗ 106 cells/mL (OD750nm approx. 0.03.)
3.4. Sample Staining
- 3.
- To stain 2 mL of sample solution, we calculated the volume of dye stock solution to be used, corresponding to the final concentration for every run, according to the CCD design (Table 2).
- ⚠ CRITICAL STEP: before use, allow the tubes containing the stock solution to warm at room temperature and then briefly centrifuge before every use to mix the DMSO with the dye.
- 4.
- After staining, put the Eppendorf tubes in the Thermomixer, at 350 rpm, for continuous mixing, with an incubation time and staining temperature according to the CCD design (Table 2).
- ⚠ CRITICAL STEP: at the end of the incubation time and before cytometric measurement, centrifuge briefly to obtain a homogeneous mixture.
3.5. Cytometric Measurements
- 5.
- Chlorophyll fluorescence was measured in the FL3 channel (675 ± 50 nm) and dye fluorescence was measured in the FL1 channel (520 ± 20 nm), as were forward scattered (FSC) and side scattered (SSC) light.
- ⚠ CRITICAL STEP: before starting the measurements, set the flow rate of the cytometer to approximately 103–2 ∗ 103 events per second, to be sure to detect all the cells and avoid detecting duplicates.
3.6. Cytometric Data Analyses
- 6.
- For each detected event, numerical values for its chlorophyll fluorescence (FL3), dye fluorescence (FL1) and forward (FSC) and side (SSC) scattering were recorded in (fcs) files. FCS ExpressTM software (De Novo Software, Pasadena, CA, USA) was used to process these collected data.
- 7.
- ⚠ CRITICAL STEP: cytometric analysis of at least a triplicate of the microalgae sample without staining is required in order to estimate the microalgae population that will serve as a control. In this study, the microalgae control population represented 97% ± 0.3 (values were calculated based on a triplicate).
3.6.1. Damaged Cells
- 8.
3.6.2. Staining Efficiency
- 9.
- The staining efficiency was determined after staining by estimating what percentage of the undamaged microalgae population have cells with stained DNA (Figure 2b).
3.7. Statistical Analysis
4. Results and Discussion
4.1. DOE—Output Responses and Model Fitting
4.2. Analysis of Variance (ANOVA)
4.3. Protocol Optimization and Validation
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Independent Variables | Symbols | Unit | −1 Level | 0 Level | +1 Level |
---|---|---|---|---|---|
Dye Concentration | A | X *–µM ** | 1.21 *–0.6 ** | 2.25 *–0.75 ** | 3.3 *–0.9 ** |
Incubation Time | B | min | 07 | 10 | 13 |
Staining Temperature | C | °C | 22 | 25 | 28 |
Run | Dye Concentration [X *–µM **] | Time [min] | Temperature [°C] | Staining Efficiency [%] | Damaged Cells [%] | |||||
---|---|---|---|---|---|---|---|---|---|---|
* | ** | * | ** | * | ** | * | ** | * | ** | |
01 | 2.25 | 0.6 | 10 | 7 | 20 | 22 | 99.83 | 99.76 | 51.29 | 18.19 |
02 | 3.3 | 0.75 | 13 | 10 | 28 | 25 | 99.79 | 99.71 | 42.37 | 16.74 |
03 | 1.21 | 0.75 | 7 | 10 | 28 | 20 | 99.84 | 99.76 | 38.32 | 22.1 |
04 | 2.25 | 0.75 | 10 | 10 | 30 | 25 | 99.81 | 99.87 | 36.24 | 23.94 |
05 | 1.21 | 0.75 | 13 | 10 | 22 | 25 | 99.87 | 99.85 | 40.21 | 27.12 |
06 | 2.25 | 0.9 | 10 | 7 | 25 | 28 | 99.65 | 99.79 | 26.74 | 24.07 |
07 | 4 | 0.9 | 10 | 7 | 25 | 22 | 99.82 | 99.81 | 70.52 | 28.41 |
08 | 3.3 | 1 | 7 | 10 | 28 | 25 | 99.8 | 99.86 | 44.53 | 32.71 |
09 | 2.25 | 0.75 | 10 | 15 | 25 | 25 | 99.76 | 99.85 | 52.57 | 23.33 |
10 | 2.25 | 0.9 | 15 | 13 | 25 | 28 | 99.8 | 99.77 | 44.99 | 18.57 |
11 | 1.21 | 0.9 | 7 | 13 | 22 | 22 | 99.79 | 99.8 | 34.21 | 25.08 |
12 | 1.21 | 0.6 | 13 | 7 | 28 | 28 | 99.74 | 99.72 | 20.92 | 11.7 |
13 | 2.25 | 0.75 | 5 | 5 | 25 | 25 | 99.78 | 99.81 | 40.55 | 24.3 |
14 | 2.25 | 0.5 | 10 | 10 | 25 | 25 | 99.72 | 99.78 | 32.79 | 13.89 |
15 | 3.3 | 0.6 | 13 | 13 | 22 | 28 | 99.85 | 99.71 | 53.79 | 9.6 |
16 | 2.25 | 0.75 | 10 | 10 | 25 | 25 | 99.83 | 99.82 | 44.99 | 19.99 |
17 | 3.3 | 0.75 | 7 | 10 | 22 | 30 | 99.84 | 99.75 | 53.25 | 14.35 |
18 | 2.25 | 0.75 | 10 | 10 | 25 | 25 | 99.84 | 99.83 | 48.41 | 22.19 |
19 | 2.25 | 0.6 | 10 | 13 | 25 | 22 | 99.79 | 99.75 | 43.85 | 14.31 |
20 | 0.5 | 0.75 | 10 | 10 | 25 | 25 | 99.67 | 99.82 | 14.16 | 19.8 |
Damaged Cells | ||||||
---|---|---|---|---|---|---|
Source | SYBR Green I | |||||
SS | DF | MS | F-Value | p-Value | Adjusted R2 | |
Linear * | 480.53 | 11 | 43.68 | 0.4534 | 0.8726 | 0.618 |
2FI | 380.35 | 8 | 47.54 | 0.4935 | 0.8212 | 0.579 |
Quadratic | 378.11 | 5 | 75.62 | 0.7849 | 0.6015 | 0.454 |
Cubic | 13.99 | 1 | 13.99 | 0.1453 | 0.7188 | 0.476 |
Pure error | 481.7 | 5 | 96.34 | |||
SYTO9 | ||||||
Linear * | 92.49 | 11 | 8.41 | 0.6395 | 0.7511 | 0.719 |
2FI | 91.44 | 8 | 11.43 | 0.8693 | 0.5914 | 0.656 |
Quadratic | 40.7 | 5 | 8.14 | 0.619 | 0.6942 | 0.6977 |
Cubic | 29.67 | 1 | 29.67 | 2.26 | 0.1934 | 0.548 |
Pure error | 65.74 | 5 | 13.15 |
Source | SYBR Green I | SYTO9 | ||
---|---|---|---|---|
F-Value | p-Value | F-Value | p-Value | |
Model | 11.26 | 0.0003 * | 17.21 | <0.0001 * |
A—Dye Concentration | 29.28 | <0.0001 * | 40.53 | <0.0001 * |
B—Incubation Time | 0.0375 | 0.8488 | 2 | 0.1763 |
C—Staining Temperature | 4.48 | 0.0504 | 9.11 | 0.0082 * |
Lack of Fit | 0.4534 | 0.8726 | 0.6395 | 0.7511 |
Staining Dye | Responses | |||||||
---|---|---|---|---|---|---|---|---|
Optimum Conditions | Desirability | Damaged Cells [%] | Staining Efficiency [%] | |||||
Dye Concentration | Incubation Time | Staining Temperature | Predicted | Actual | Predicted | Actual | ||
SYBR Green I | 0.5 X | 5 min | 25 °C | 1 | 23.32 | 13.75 | 99.77 | 99.60 |
SYTO9 | 0.5 µM | 5 min | 25 °C | 0.93 | 13.43 | 12.86 | 99.74 | 99.80 |
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Ihadjadene, Y.; Walther, T.; Krujatz, F. Optimized Protocol for Microalgae DNA Staining with SYTO9/SYBR Green I, Based on Flow Cytometry and RSM Methodology: Experimental Design, Impacts and Validation. Methods Protoc. 2022, 5, 76. https://doi.org/10.3390/mps5050076
Ihadjadene Y, Walther T, Krujatz F. Optimized Protocol for Microalgae DNA Staining with SYTO9/SYBR Green I, Based on Flow Cytometry and RSM Methodology: Experimental Design, Impacts and Validation. Methods and Protocols. 2022; 5(5):76. https://doi.org/10.3390/mps5050076
Chicago/Turabian StyleIhadjadene, Yob, Thomas Walther, and Felix Krujatz. 2022. "Optimized Protocol for Microalgae DNA Staining with SYTO9/SYBR Green I, Based on Flow Cytometry and RSM Methodology: Experimental Design, Impacts and Validation" Methods and Protocols 5, no. 5: 76. https://doi.org/10.3390/mps5050076