Optimization of the Hemolysis Assay for the Assessment of Cytotoxicity
<p>OD measurements at 405 nm (Y-axis) of free hemoglobin in a 1% erythrocyte solution originating from a mouse, rat, rabbit, and human, incubated for 60 min at 37 °C with PBS (negative control), 10% Triton X-100 (positive control), or AMPs 1, 2 or 3 (at concentrations of 100 µM). Average values from three experimental replicates, each containing two technical replicates, are presented with error bars (SD) included in plots. Significantly different data as defined from unpaired <span class="html-italic">t</span>-test is indicated by asterisks for comparison of human and rabbit samples (<span class="html-italic">p</span>-values: * < 0.05 *** < 0.001 **** < 0.0001 ns: non-significant). See <a href="#app1-ijms-24-02914" class="html-app">Table S1</a> for <span class="html-italic">p</span>-values from comparison of all species.</p> "> Figure 2
<p>OD measurements at 405 nm (Y-axis) of free hemoglobin in 1% erythrocyte solutions originating from 10 different human individuals, as well as blood pooled from all individuals, incubated for 60 min at 37 °C with PBS (negative control), 10% Triton X-100 (positive control), dH<sub>2</sub>O, or AMPs 1, 2, or 3 (100 µM). Average values from three experimental replicates, each containing two technical replicates, are presented with error bars (SD) included in plots.</p> "> Figure 3
<p>OD measurements at 405 nm (Y-axis) of free hemoglobin in 1%, 2%, and 5% erythrocyte (EC) solution of pooled blood originating from 10 different human individuals, incubated for 60 min at 37 °C with PBS (negative control), 10% Triton X-100 (positive control), dH<sub>2</sub>O, or AMPs 1, 2, or 3 (100 µM). Average values from three experimental replicates, each containing two technical replicates, are presented with error bars (SD) and included in plots.</p> "> Figure 4
<p>OD measurements at 405 nm (Y-axis) of free hemoglobin in a 1% human erythrocyte solution incubated for 15, 30, 60, 90 or 120 min at 37 °C with PBS (negative control), 10% Triton X-100 (positive control), dH<sub>2</sub>O or AMPs 1, 2 or 3 (100 µM). Average values from three experimental replicates, each containing two technical replicates, are presented with error bars (SD) included in plots.</p> "> Figure 5
<p>OD measurements at 405 nm (Y-axis) of free hemoglobin in a 1% erythrocyte solution sourced from a mouse, rat, rabbit, and human treated with different concentrations of Triton X-100 (<b>A</b>), Tween (<b>B</b>), and SDS (<b>C</b>), as well as ACS or dH<sub>2</sub>O (<b>D</b>). Erythrocytes were incubated for 60 min at 37 °C. Average values from three experimental replicates, each containing two technical replicates, are presented with error bars (SD) included in plots. Significantly different data as defined from unpaired <span class="html-italic">t</span>-test is indicated by asterisks (<span class="html-italic">p</span>-values: * < 0.05 ** < 0.01 *** < 0.001 **** < 0.0001 ns: non-significant).</p> "> Figure 6
<p>OD measurements of free hemoglobin in human whole blood at 405 nm (<b>A</b>), 530 nm (<b>B</b>), or 570 nm (<b>C</b>). Samples were treated with PBS (negative control), AMPs 1, 2, 3, melittin, or polymyxin B (all at 100 μM), as well as with dH<sub>2</sub>O, ACS, or different concentrations of Triton X-100, Tween, or SDS. All samples were incubated for 60 min at 37 °C. Average values from three experimental replicates, each containing two technical replicates, are presented with error bars (SD) included in plots.</p> "> Figure 7
<p>OD measurements of free hemoglobin in 1% washed human erythrocytes at 405 nm (<b>A</b>), 530 nm (<b>B</b>), or 570 nm (<b>C</b>). Samples were treated with PBS (negative control), AMPs 1, 2, 3, melittin or polymyxin B (all at 100 μM), as well as with dH<sub>2</sub>O, ACS, or different concentrations of Triton X-100, Tween, or SDS. All samples were incubated for 60 min at 37 °C. Average values from three experimental replicates, each containing two technical replicates, are presented with error bars (SD) included in plots.</p> "> Figure 8
<p>OD measurements at 405 nm (Y-axis) of free hemoglobin in a 1% human erythrocyte solution incubated with PBS, dH<sub>2</sub>O, ACS, Triton X-100, SDS, Tween, AMPs 1, 2, or 3 (100 μM), or polymyxin B (100 μM) for 60 min at 37 °C in polystyrene (black bars) or polypropylene (red bars) tubes. Average values from three experimental replicates, each containing two technical replicates, are presented with error bars (SD) included in plots. Significantly different data as defined from paired <span class="html-italic">t</span>-test is indicated by asterisks (ns: non-significant).</p> "> Figure 9
<p>OD measurements at 405 nm (Y-axis) of free hemoglobin in a 1% human erythrocyte solution incubated with PBS, dH<sub>2</sub>O, ACS, Triton X-100, SDS, Tween, AMPs 1, 2, or 3 (100 μM), or polymyxin B (100 μM) for 60 min at 37 °C using high (500 μL: black bars) or low (100 μL; red bars) volumes. Average values from three experimental replicates, each containing two technical replicates, are presented with error bars (SD) included in plots. Significantly different data as defined from paired <span class="html-italic">t</span>-test is indicated by asterisks (<span class="html-italic">p</span>-values: * < 0.05 ns: non-significant).</p> ">
Abstract
:1. Introduction
2. Results
2.1. Species-Dependent Effects on Hemolysis
2.2. Blood Drawn from Different Human Individuals Appears to Respond to Hemolytic Agents to a Similar Extent
2.3. The Erythrocyte Concentration in Samples Affects Measurements of Free Hemoglobin
2.4. Sample Incubation Time Affects the Degree of Hemolysis
2.5. Large Variations in Hemolytic Response to Different Detergents Used as Positive Controls
2.6. Use of Whole Blood vs. Washed Erythrocytes in Hemolysis Assays: Choice of Wavelength and Effect on Hemolysis
2.7. Plasticware Used for Incubation
2.8. Downscaling of Sample Volumes to 96-Well Format Retains Hemolysis Ratio Data Quality
3. Discussion
3.1. Choosing Species Origin of Erythrocytes
3.2. Washed Erythrocytes vs. Whole Blood
3.3. Erythrocyte Concentration and Incubation Time
3.4. Choosing the Most Efficient Detergent as a Positive Control
3.5. Downscaling of Experiments to 96-Well Plates
3.6. Other Remarks
- Triton X-100 (Sigma-Aldrich, Saint-Louis, MO, USA, T8787)
- Phosphate-buffered Saline (PBS) pH~7
- LH Lithium Heparin tubes (4 mL; Greiner Bio-One, Frickenhausen, Germany, 454029) or Sodium Citrate 3.2% tubes (3 mL; Greiner Bio-One, Frickenhausen, Germany, 454334)
- 96-well Polypropylene PCR plate (VWR, Radnor, PA, USA, 82006-636 or similar). For incubation step.
- 96-well plate with flat, transparent bottom (Anicrin, Moglianese, Italy, A013418 or similar). For OD measurements in the plate reader.
- Collect blood in heparin or sodium citrate tubes and immediately centrifuge at 1700× g for 5 min. Avoid using needles above 23 G in order to minimize pre-analyte hemolysis.
- Remove the supernatant by aspiration and wash the erythrocytes by adding 2 mL of PBS pH~7. Centrifuge at 1700× g for 5 min. Repeat the washing step three times or until supernatant is clear.
- Remove supernatant and dilute the erythrocyte pellet 1:100 in PBS pH~7 to obtain a 1% erythrocyte suspension.
- Mix 50 µL of the 1% erythrocyte suspension with 50 µL of test compound in a 96-well polypropylene plate with conical wells (PCR plate). The conical shape makes it easier to pipette the supernatant in the next step. Use 10% Triton X-100 as a positive control and PBS pH~7 as a negative control in identical volumes as test compounds. NB: 10% Triton X-100 solution should be made by weighing (w/v) to obtain an accurate concentration.
- Incubate the plate at 37 °C for 60 min.
- Centrifuge the plate at 1700× g for 5 min.
- Transfer 50 µL of the supernatant to a transparent, flat-bottom 96-well plate and measure absorption at 405 nm in a plate reader.
4. Materials and Methods
4.1. Blood Collection
4.2. Antimicrobial Peptides
4.3. Preparation of Detergent Solutions for Positive Controls
4.4. Cell Counting
4.5. Flow Cytometry of Erythrocytes Treated with Live/Dead Stain
4.6. General Procedure of the Hemolysis Assays
4.7. Data Management
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Species | AMP 1 | AMP 2 | AMP 3 |
---|---|---|---|
Mouse | 29.2 ± 32 | 14.9 ± 14.9 | 50.1 ± 14.4 |
Rat | 12.9 ± 1.7 | 5.2 ± 1.9 | 29.5 ± 8.2 |
Rabbit | 7.1 ± 1.1 | 4.6 ± 0.7 | 53.2 ± 7.4 |
Human | 22.3 ± 4 | 8.5 ± 1.4 | 37 ± 5.1 |
Human Ind. | dH2O | AMP 1 | AMP 2 | AMP 3 |
---|---|---|---|---|
# 1 | 38 ± 12.3 | 21.9 ± 4 | 8.2 ± 1.3 | 36.4 ± 5 |
# 2 | 57.7 ± 12.2 | 23 ± 2.8 | 10 ± 0.7 | 39.3 ± 6 |
# 3 | 50.8 ± 8.8 | 23.7 ± 2.1 | 9.8 ± 0.8 | 49.4 ± 8.5 |
# 4 | 47.2 ± 5.6 | 21.3 ± 3 | 10.4 ± 0.9 | 43.6 ± 4.2 |
# 5 | 38.8 ± 12.1 | 19.7 ± 3.7 | 11 ± 1 | 38.3 ± 7.3 |
# 6 | 36.2 ± 10.8 | 19.6 ± 4.7 | 6.4 ± 0.7 | 45.7 ± 6.4 |
# 7 | 49.6 ± 15.2 | 18.8 ± 4 | 8.9 ± 0.8 | 37.7 ± 6.9 |
# 8 | 49.4 ± 16.9 | 21.7 ± 8.6 | 9.1 ± 1.1 | 45.6 ± 9.2 |
# 9 | 49.1 ± 22.5 | 22.9 ± 6.8 | 9.6 ± 1 | 42.1 ± 4.7 |
# 10 | 39.4 ± 10.7 | 25.1 ± 13 | 9.8 ± 0.7 | 50.3 ± 4.6 |
Pooled | 32.6 ± 7.3 | 22.7 ± 6.6 | 12.4 ± 3.1 | 45.4 ± 4.8 |
Calculated Hemolysis (%) | dH2O | AMP 1 | AMP 2 | AMP 3 |
---|---|---|---|---|
1% erythrocytes | 32.6 ± 7.3 | 22.7 ± 6.6 | 12.4 ± 3.1 | 45.4 ± 4.8 |
2% erythrocytes | 27.1 ± 2.7 | 13.7 ± 4.3 | 5.6 ± 0.8 | 41.1 ± 3.2 |
5% erythrocytes | 22.7 ± 9 | 10.8 ± 3.2 | 5.0 ± 3.3 | 45.7 ± 8.2 |
Incubation Time | dH2O | AMP 1 | AMP 2 | AMP 3 |
---|---|---|---|---|
15 min | 32.8 ± 12.1 | 18.5 ± 2.3 | 7.8 ± 1.1 | 54.8 ± 8.2 |
30 min | 34.8 ± 12.7 | 20.9 ± 4 | 7.4 ± 1.2 | 56.6 ± 7.4 |
60 min | 37.1 ±14.2 | 26.6 ± 4.7 | 7.8 ± 1.3 | 64.6 ± 6.8 |
90 min | 40.1 ±13.9 | 26.9 ± 6 | 8 ± 0.8 | 63 ± 4.5 |
120 min | 47 ± 13.6 | 30.6 ± 10.6 | 8.6 ± 1.4 | 74.3 ± 21.1 |
Wavelength | dH2O | ACS | AMP 1 | AMP 2 | AMP 3 | Melittin | PMB |
---|---|---|---|---|---|---|---|
405 nm | 98.2 ± 0.4 | 97.8 ± 1.3 | 3.3 ± 3.2 | 0.4 ± 0.2 | 15.5 ± 7.1 | 98.3 ± 1.4 | 3.1 ± 1.1 |
530 nm | 38.4 ± 8.5 | 28.1 ± 4.5 | 0.4 ± 0.7 | 0.2 ± 0.4 | 2.7 ± 1.4 | 36.6 ± 8.7 | 3 ± 2.4 |
570 nm | 62.8 ± 1.4 | 45.1 ± 7.7 | 0.4 ± 1.2 | −0.1 ± 0.3 | 3.8 ± 2.1 | 54 ± 15.5 | 4.5 ± 1.8 |
Wavelength | dH2O | ACS | AMP 1 | AMP 2 | AMP 3 | Melittin | PMB |
---|---|---|---|---|---|---|---|
405 nm | 73.9 ± 7.5 | 94.7 ± 14.4 | 27.7 ± 4.6 | 8.2 ± 1.4 | 73.5 ± 14.4 | 84.4 ± 8.1 | 4.4 ± 0.3 |
530 nm | 67.6 ± 8.7 | 88.5 ± 14.3 | 29.2 ± 8 | 5.2 ± 3 | 68.4 ± 15 | 78.4 ± 4.3 | 3.0 ± 4.7 |
570 nm | 71.2 ± 10.3 | 89.7 ± 13.4 | 25.3 ± 5.9 | 4.8 ± 6.6 | 65.7 ± 11.6 | 79.8 ± 8.2 | 1.3 ± 4.7 |
dH2O | ACS | AMP 1 | AMP 2 | AMP 3 | PMB | |
---|---|---|---|---|---|---|
Polystyrene | 43.1 ± 18.6 | 71.2 ± 13.4 | 23.8 ± 4.3 | 9.7 ± 3.4 | 66.7 ± 12.7 | 6.1 ± 3.1 |
Polypropylene | 29.5 ± 6 | 52.3 ± 9.2 | 19 ± 0.7 | 8.2 ± 2.6 | 57.5 ± 13.2 | 3.2 ± 1 |
dH2O | ACS | AMP 1 | AMP 2 | AMP 3 | PMB | |
---|---|---|---|---|---|---|
100 μL | 43.1 ± 18.6 | 71.2 ± 13.4 | 23.8 ± 4.3 | 9.7 ± 3.4 | 66.7 ± 12.7 | 6.1 ± 3.1 |
500 μL | 31.4 ± 10.7 | 56.7 ± 8.8 | 21.9 ± 1.2 | 8.8 ± 3.9 | 72.7 ± 10.4 | 6.1 ± 2.8 |
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Sæbø, I.P.; Bjørås, M.; Franzyk, H.; Helgesen, E.; Booth, J.A. Optimization of the Hemolysis Assay for the Assessment of Cytotoxicity. Int. J. Mol. Sci. 2023, 24, 2914. https://doi.org/10.3390/ijms24032914
Sæbø IP, Bjørås M, Franzyk H, Helgesen E, Booth JA. Optimization of the Hemolysis Assay for the Assessment of Cytotoxicity. International Journal of Molecular Sciences. 2023; 24(3):2914. https://doi.org/10.3390/ijms24032914
Chicago/Turabian StyleSæbø, Ingvill Pedersen, Magnar Bjørås, Henrik Franzyk, Emily Helgesen, and James Alexander Booth. 2023. "Optimization of the Hemolysis Assay for the Assessment of Cytotoxicity" International Journal of Molecular Sciences 24, no. 3: 2914. https://doi.org/10.3390/ijms24032914