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Analysis of damage-associated molecular pattern molecules due to electroporation of cells in vitro Cover

Analysis of damage-associated molecular pattern molecules due to electroporation of cells in vitro

Radiology and Oncology
Volume 54 (2020): Issue 3 (September 2020)
By: Tamara Polajzer,  Tomaz Jarm and  Damijan Miklavcic  
Open Access
|Jul 2020

Figures & Tables

Figure 1

Application of 500 V pulses. Blue line shows voltage and red line shows current. Due to sequencing, all eight pulses are in one picture, separated by //.
Application of 500 V pulses. Blue line shows voltage and red line shows current. Due to sequencing, all eight pulses are in one picture, separated by //.

Figure 2

Release of adenosine triphosphate (ATP) as a function of electric pulse amplitude determined by fluorescent assay. Two-time points after electroporation were assessed. Permeabilization and survival curves are also presented. Black and green asterisks (*) indicate statistically significant differences between the samples at different voltages and the corresponding control at 0 V (one-way analysis of variance [ANOVA] followed by Holm-Sidak post-hoc test, (p < 0.05) and within the pair of samples at different voltages (t-test, p < 0.05), respectively).
Release of adenosine triphosphate (ATP) as a function of electric pulse amplitude determined by fluorescent assay. Two-time points after electroporation were assessed. Permeabilization and survival curves are also presented. Black and green asterisks (*) indicate statistically significant differences between the samples at different voltages and the corresponding control at 0 V (one-way analysis of variance [ANOVA] followed by Holm-Sidak post-hoc test, (p < 0.05) and within the pair of samples at different voltages (t-test, p < 0.05), respectively).

Figure 3

Release of adenosine triphosphate (ATP), as a function of electric pulse amplitude determined by luminescence assay. Two-time points after electroporation were assessed. Permeabilization and survival curves are also presented. Black and green asterisks (*) indicate statistically significant differences between the samples at different voltages and the corresponding control at 0 V (one-way analysis of variance [ANOVA] followed by Holm-Sidak post-hoc test, (p < 0.05) and within the pair of samples at different voltages (t-test, p < 0.05), respectively).
Release of adenosine triphosphate (ATP), as a function of electric pulse amplitude determined by luminescence assay. Two-time points after electroporation were assessed. Permeabilization and survival curves are also presented. Black and green asterisks (*) indicate statistically significant differences between the samples at different voltages and the corresponding control at 0 V (one-way analysis of variance [ANOVA] followed by Holm-Sidak post-hoc test, (p < 0.05) and within the pair of samples at different voltages (t-test, p < 0.05), respectively).

Figure 4

Externalization of calreticulin as a function of electric pulse amplitude. Two-time points after electroporation were assessed. Permeabilization and survival (MTS) curves are also presented. Survival detected by propidium iodide (PI) protocol is normalized to control and presented with red (4 hours after pulse treatment) and orange (24 hours after pulse treatment) line. Approximate baseline of calreticulin is presented with -----. Black and green asterisks (*) indicate statistically significant differences between the samples at different voltages and the corresponding control at 0 V (one-way analysis of variance [ANOVA] followed by Holm-Sidak posthoc test, (p < 0.05) and within the pair of samples at different voltages (t-test, p < 0.05), respectively).
Externalization of calreticulin as a function of electric pulse amplitude. Two-time points after electroporation were assessed. Permeabilization and survival (MTS) curves are also presented. Survival detected by propidium iodide (PI) protocol is normalized to control and presented with red (4 hours after pulse treatment) and orange (24 hours after pulse treatment) line. Approximate baseline of calreticulin is presented with -----. Black and green asterisks (*) indicate statistically significant differences between the samples at different voltages and the corresponding control at 0 V (one-way analysis of variance [ANOVA] followed by Holm-Sidak posthoc test, (p < 0.05) and within the pair of samples at different voltages (t-test, p < 0.05), respectively).

Figure 5

Release of RNA as a function of electric pulse amplitude. Three-time points after electroporation were assessed. Permeabilization and survival curves are also presented. Approximate baseline of RNA is presented with -----. Black and green asterisks (*) indicate statistically significant differences between the samples at different voltages and the corresponding control at 0 V (one-way analysis of variance [ANOVA] followed by Holm-Sidak post-hoc test, (p < 0.05) and within the pair of samples at different voltages (t-test, p < 0.05), respectively).
Release of RNA as a function of electric pulse amplitude. Three-time points after electroporation were assessed. Permeabilization and survival curves are also presented. Approximate baseline of RNA is presented with -----. Black and green asterisks (*) indicate statistically significant differences between the samples at different voltages and the corresponding control at 0 V (one-way analysis of variance [ANOVA] followed by Holm-Sidak post-hoc test, (p < 0.05) and within the pair of samples at different voltages (t-test, p < 0.05), respectively).

Figure 6

Release of DNA as a function of electric pulse amplitude. Three-time points after electroporation were assessed. Permeabilization and survival curves are also presented. Approximate baseline of DNA is presented with -----. Black and green asterisks (*) indicate statistically significant differences between the samples at different voltages and the corresponding control at 0 V (one-way analysis of variance [ANOVA] followed by Holm-Sidak post-hoc test, (p < 0.05) and within the pair of samples at different voltages (t-test, p < 0.05) respectively.
Release of DNA as a function of electric pulse amplitude. Three-time points after electroporation were assessed. Permeabilization and survival curves are also presented. Approximate baseline of DNA is presented with -----. Black and green asterisks (*) indicate statistically significant differences between the samples at different voltages and the corresponding control at 0 V (one-way analysis of variance [ANOVA] followed by Holm-Sidak post-hoc test, (p < 0.05) and within the pair of samples at different voltages (t-test, p < 0.05) respectively.

Figure 7

Release of uric acid as a function of electric pulse amplitude. Amount of uric acid was analysed 24 hours after pulse treatment in supernatant. Permeabilization and survival curves are also presented. No statistical difference was detected.
Release of uric acid as a function of electric pulse amplitude. Amount of uric acid was analysed 24 hours after pulse treatment in supernatant. Permeabilization and survival curves are also presented. No statistical difference was detected.

Correlation (R) between permeabilization and release of damage-associated molecular pattern molecules (DAMPs) after pulse treatment_ Investigated time points for each molecule are present in the bottom row_ Correlation was evaluated with Pearson correlation coefficient and permeabilization was analyzed by propidium iodide (PI) assay 3 minutes after pulse treatment

R vs permeabilization (PI)
MTS-0.680
ATP0.7040.728 (L)/0.594 (F)-0.695 (F)
DNA0.5710.689NS
RNA0.5840.696NS
CRT0.5350.556
uric acidNS
time after points EP3 min15 min30 min4 h24 h

Correlation (R) between survival and release of damage-associated molecular pattern molecules (DAMPs) after pulse treatment_ Investigated time points for each molecule are presented in the bottom row_ Correlation was evaluated with Pearson correlation coefficient and survival was analyzed via MTS assay 24 hours after pulse treatment

R vs. survival (MTS)
PI-0.680
ATP-0.947 (L)-0.964 (L)/-0.864 (F)0.888 (F)
DNA-0.935-0.919-0.928
RNA-0.909-0.909-0.919
CRT-0.801-0.946
uric acidNS
time after points EP3 min15 min30 min4 h24 h
DOI: https://doi.org/10.2478/raon-2020-0047 | Journal eISSN: 1581-3207 | Journal ISSN: 1318-2099
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Language: English
Page range: 317 - 328
Submitted on: Jun 18, 2020
|
Accepted on: Jul 7, 2020
|
Published on: Jul 29, 2020
Published by: Association of Radiology and Oncology
In partnership with: Paradigm Publishing Services
Publication frequency: 4 issues per year
Keywords:
electroporation,
pulsed electric field treatment,
damage-associated molecular pattern molecules,
immunogenic cell death,
electrochemotherapy
Related subjects:
Medicine,
Clinical medicine,
Internal medicine,
Haematology, oncology,
Radiology

© 2020 Tamara Polajzer, Tomaz Jarm, Damijan Miklavcic, published by Association of Radiology and Oncology
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

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