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The 2D cell monolayer cryopreservation using HPF and NPF. a) Representative PI/Hoechst merged images of the live control (no treatment), dead control (70% ethanol treatment), and freezing/thawing groups. Dead cells were identified by PI staining, while live cells were defined as Hoechst-positive and PI-negative (shown in magenta and blue, respectively). Scale bars, 500 μm. b) Live cell number (normalized by control group) of the live control and freezing/thawing groups. Six different CPAs (all containing 20% dextran) were used for HPF and NPF. Cell counting was based on PI/Hoechst staining. <t>c)</t> <t>WST-8</t> absorbance (normalized by control group) of the live control and freezing/thawing groups. To account for potential growth arrest following cryopreservation, for the freezing/thawing groups, WST-8 was measured on day 1 postthawing, while for the live control, it was measured on day 0. d) Cell growth of the live control and freezing/thawing groups. To account for potential growth arrest following cryopreservation, WST-8 assays were performed on day 1 and day 5 postthawing for the freezing/thawing groups and on day 0 and day 4 for the live control. n = 4 independent experiments. Statistical analysis was performed using two-way ANOVA with Tukey's post hoc. P -values for key pairwise comparisons are shown.
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The 2D cell monolayer cryopreservation using HPF and NPF. a) Representative PI/Hoechst merged images of the live control (no treatment), dead control (70% ethanol treatment), and freezing/thawing groups. Dead cells were identified by PI staining, while live cells were defined as Hoechst-positive and PI-negative (shown in magenta and blue, respectively). Scale bars, 500 μm. b) Live cell number (normalized by control group) of the live control and freezing/thawing groups. Six different CPAs (all containing 20% dextran) were used for HPF and NPF. Cell counting was based on PI/Hoechst staining. <t>c)</t> <t>WST-8</t> absorbance (normalized by control group) of the live control and freezing/thawing groups. To account for potential growth arrest following cryopreservation, for the freezing/thawing groups, WST-8 was measured on day 1 postthawing, while for the live control, it was measured on day 0. d) Cell growth of the live control and freezing/thawing groups. To account for potential growth arrest following cryopreservation, WST-8 assays were performed on day 1 and day 5 postthawing for the freezing/thawing groups and on day 0 and day 4 for the live control. n = 4 independent experiments. Statistical analysis was performed using two-way ANOVA with Tukey's post hoc. P -values for key pairwise comparisons are shown.
Wst 8 Solution, supplied by Dojindo Labs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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The 2D cell monolayer cryopreservation using HPF and NPF. a) Representative PI/Hoechst merged images of the live control (no treatment), dead control (70% ethanol treatment), and freezing/thawing groups. Dead cells were identified by PI staining, while live cells were defined as Hoechst-positive and PI-negative (shown in magenta and blue, respectively). Scale bars, 500 μm. b) Live cell number (normalized by control group) of the live control and freezing/thawing groups. Six different CPAs (all containing 20% dextran) were used for HPF and NPF. Cell counting was based on PI/Hoechst staining. <t>c)</t> <t>WST-8</t> absorbance (normalized by control group) of the live control and freezing/thawing groups. To account for potential growth arrest following cryopreservation, for the freezing/thawing groups, WST-8 was measured on day 1 postthawing, while for the live control, it was measured on day 0. d) Cell growth of the live control and freezing/thawing groups. To account for potential growth arrest following cryopreservation, WST-8 assays were performed on day 1 and day 5 postthawing for the freezing/thawing groups and on day 0 and day 4 for the live control. n = 4 independent experiments. Statistical analysis was performed using two-way ANOVA with Tukey's post hoc. P -values for key pairwise comparisons are shown.
Wst 8 Reagent Cell, supplied by Dojindo Labs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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The 2D cell monolayer cryopreservation using HPF and NPF. a) Representative PI/Hoechst merged images of the live control (no treatment), dead control (70% ethanol treatment), and freezing/thawing groups. Dead cells were identified by PI staining, while live cells were defined as Hoechst-positive and PI-negative (shown in magenta and blue, respectively). Scale bars, 500 μm. b) Live cell number (normalized by control group) of the live control and freezing/thawing groups. Six different CPAs (all containing 20% dextran) were used for HPF and NPF. Cell counting was based on PI/Hoechst staining. c) WST-8 absorbance (normalized by control group) of the live control and freezing/thawing groups. To account for potential growth arrest following cryopreservation, for the freezing/thawing groups, WST-8 was measured on day 1 postthawing, while for the live control, it was measured on day 0. d) Cell growth of the live control and freezing/thawing groups. To account for potential growth arrest following cryopreservation, WST-8 assays were performed on day 1 and day 5 postthawing for the freezing/thawing groups and on day 0 and day 4 for the live control. n = 4 independent experiments. Statistical analysis was performed using two-way ANOVA with Tukey's post hoc. P -values for key pairwise comparisons are shown.

Journal: PNAS Nexus

Article Title: A proof-of-concept study on high-pressure freezing for cryopreservation

doi: 10.1093/pnasnexus/pgag065

Figure Lengend Snippet: The 2D cell monolayer cryopreservation using HPF and NPF. a) Representative PI/Hoechst merged images of the live control (no treatment), dead control (70% ethanol treatment), and freezing/thawing groups. Dead cells were identified by PI staining, while live cells were defined as Hoechst-positive and PI-negative (shown in magenta and blue, respectively). Scale bars, 500 μm. b) Live cell number (normalized by control group) of the live control and freezing/thawing groups. Six different CPAs (all containing 20% dextran) were used for HPF and NPF. Cell counting was based on PI/Hoechst staining. c) WST-8 absorbance (normalized by control group) of the live control and freezing/thawing groups. To account for potential growth arrest following cryopreservation, for the freezing/thawing groups, WST-8 was measured on day 1 postthawing, while for the live control, it was measured on day 0. d) Cell growth of the live control and freezing/thawing groups. To account for potential growth arrest following cryopreservation, WST-8 assays were performed on day 1 and day 5 postthawing for the freezing/thawing groups and on day 0 and day 4 for the live control. n = 4 independent experiments. Statistical analysis was performed using two-way ANOVA with Tukey's post hoc. P -values for key pairwise comparisons are shown.

Article Snippet: Metabolic activity was assessed 24 h postthawing using the WST-8 assay kit (Cell Counting Kit-8, Dojindo).

Techniques: Control, Staining, Cell Counting

Cell spheroid cryopreservation using HPF and NPF. a) Confocal and bright-field images of cell spheroids from the live control and freezing/thawing groups. Arrows highlight representative cells exhibiting nuclear fragmentation and nuclear condensation. Scale bar, 50 μm. b) Cell spheroid diameter of live control and freezing/thawing groups. Feret's diameters of the cell spheroids in (a) were measured using ImageJ. n = 9 independent experiments. c) WST-8 absorbance (normalized by control group) of the live control and freezing/thawing groups. n = 9 independent experiments. d) Confocal microscopy image of E-cadherin immunostaining in cell spheroids. For HPF and NPF groups, use 30% EG + 20% dextran as CPA. Scale bar, 50 μm. e) Fluorescence intensity of E-cadherin immunostaining of live control and freezing/thawing groups. Fluorescence intensity was quantified by ImageJ. n = 6 independent experiments. f) ATP levels (normalized by control group) of the live control and freezing/thawing groups. n = 6 independent experiments. Statistical analysis was performed using two-way ANOVA followed by Tukey's post hoc test for (b, c) and one-way ANOVA followed by Tukey's post hoc test for (e, f). P -values for key pairwise comparisons are shown.

Journal: PNAS Nexus

Article Title: A proof-of-concept study on high-pressure freezing for cryopreservation

doi: 10.1093/pnasnexus/pgag065

Figure Lengend Snippet: Cell spheroid cryopreservation using HPF and NPF. a) Confocal and bright-field images of cell spheroids from the live control and freezing/thawing groups. Arrows highlight representative cells exhibiting nuclear fragmentation and nuclear condensation. Scale bar, 50 μm. b) Cell spheroid diameter of live control and freezing/thawing groups. Feret's diameters of the cell spheroids in (a) were measured using ImageJ. n = 9 independent experiments. c) WST-8 absorbance (normalized by control group) of the live control and freezing/thawing groups. n = 9 independent experiments. d) Confocal microscopy image of E-cadherin immunostaining in cell spheroids. For HPF and NPF groups, use 30% EG + 20% dextran as CPA. Scale bar, 50 μm. e) Fluorescence intensity of E-cadherin immunostaining of live control and freezing/thawing groups. Fluorescence intensity was quantified by ImageJ. n = 6 independent experiments. f) ATP levels (normalized by control group) of the live control and freezing/thawing groups. n = 6 independent experiments. Statistical analysis was performed using two-way ANOVA followed by Tukey's post hoc test for (b, c) and one-way ANOVA followed by Tukey's post hoc test for (e, f). P -values for key pairwise comparisons are shown.

Article Snippet: Metabolic activity was assessed 24 h postthawing using the WST-8 assay kit (Cell Counting Kit-8, Dojindo).

Techniques: Control, Confocal Microscopy, Immunostaining, Fluorescence