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. 2018 Sep 4;8:13192. doi: 10.1038/s41598-018-31575-3

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Pixel co-localization between FITC (NucView488 casp3 Substrate and Calcein AM), DAPI (MitoView Blue) and TRITC (CF 594 Annexin V and EthD-1) channels from live cell images of paclitaxel (2.5 nM) + BKM120 (500 nM) treated OVK18 cells stained for triple fluorescence (A) and paclitaxel (2.5 nM) treated live-dead OVK18 cells (B) was presented. (A) Co-localization between FITC, DAPI, and TRITC channels was determined in the scatterplot (inset) of merged (channels). Image applied to all frames with a target area of whole frames (total 1428288 pixels and selected1428288 pixels). Pixel co-localization between DAPI–FITC (upper inset), DAPI–TRITC (middle inset), and FITC – TRITC (lower inset), channels with co-efficient parameters are presented (insets) in non-treated (NT; Ai) and drug-treated (Paclitaxel-BKM120; Aii) OVK18 cells. A few cells with red and green stain are marked with yellow boxes, and cells with red and blue stain are marked with violet boxes. A few cells with red, blue, and green stains are marked with red, blue, and green boxes respectively. The co-location pattern of three channels are found to be characteristically different between NT and treated group. (B) Co-localization between FITC and TRITC channels was determined in the scatterplot (inset) of merged (channels). Image applied to all frames with a target area of whole frames (total 1428288 pixels and selected1428288 pixels). Upper panel represents non-treated (NT) live-dead OVK18 cells. Pearson’s Correlation Coefficient R(r) [Whole Frame] was 0.017 (A perfect positive linear relationship, r = 1). Overlap Coefficient was 0.342. Overlap Coefficient K1 [Whole Frame] was 0.414 and K2 [Whole Frame] 0.282. Co-localization co-efficient M1(whole frame) was 0.983 M2 (whole frame) was 0.648. The lower panel represents paclitaxel-treated (Paclitaxel) live-dead OVK18 cells. Co-localization between FITC and TRITC channels was determined in the scatterplot (inset) of merged (channels). Image applied to all frames with a target area of whole frames (total 1428288 pixels and selected1428288 pixels). Pearson’s Correlation Coefficient R(r) [Whole Frame] was 0.008. (A perfect positive linear relationship, r = 1). Overlap Coefficient was 0.061. Overlap Coefficient K1 [Whole Frame] was 0.007, and K2 [Whole Frame] was 551. Co-localization co-efficient M1(whole frame) was 0.009 M2 (whole frame) was 0.552.