A high content assay to assess cellular fitness

Abstract

A universal process in experimental biology is the use of engineered cells; more often, stably or transiently transfected cells are generated for the purpose. Therefore, it is important that cell health assessment is conducted to check for stress mediated by induction of heat shock proteins (Hsps). For this purpose, we have developed an integrated platform that would enable a direct assessment of transfection efficiency (TE) combined with cellular toxicity and stress response. We make use of automated microscopy and high content analysis to extract from the same well a multiplexed readout to assess and determine optimal chemical transfection conditions. As a proof of concept, we investigated seven commercial reagents, in a matrix of dose and time, to study transfection of an EGFP DNA plasmid into HeLa cells and their consequences on health and fitness; where we scored for cellular proliferation, EGFP positive cells, and induction of Hsp10 and Hsp70 as makers of stress responses. FuGENE HD emerged as the most optimal reagent with no apparent side effects suitable for performing microtiter based miniaturized transfection for both chemical and RNAi screening. In summary, we report on a high content assay method to assess cellular overall fitness upon chemical transfection.

Figure 1. Whole well imaging on the INCA2000, quantification of cellular proliferation, and TE

(A) Whole well images are captured at 4X objective magnification on the DAPI and FITC channel for (B) Automated image analysis and quantification of Hoechst-stained nuclei (blue mask overlay) and EGFP expression (green mask overlay). Accurate quantification of the number of EGFP expressing cells relative to the total number of cells for assessment of TE is achieved by quantifying EGFP objects that overlap with a nucleus (C&D) to dismiss fluorescent artifacts and cell debris.

Figure 2. Four channel imaging at 40X objective magnification on the INCA6000 microscope

Automated laser-based imaging platform with confocal modality at 40X objective magnification of (A) Hoechst-stained nuclei, (B) anti-Hsp70/AF633 immunostaining of Hsp70, (C) Rhodamine-phalloidin staining of actin, and (D) EGFP expression.(E) Assessment of cell morphology on the fused images from A, B, C, and D; enabling visualization and potential quantification of cell stress as measured by Hsp induction levels.

Figure 2. Four channel imaging at 40X objective magnification on the INCA6000 microscope

Automated laser-based imaging platform with confocal modality at 40X objective magnification of (A) Hoechst-stained nuclei, (B) anti-Hsp70/AF633 immunostaining of Hsp70, (C) Rhodamine-phalloidin staining of actin, and (D) EGFP expression.(E) Assessment of cell morphology on the fused images from A, B, C, and D; enabling visualization and potential quantification of cell stress as measured by Hsp induction levels.

Figure 3. Automated image analysis for quantification of Hsp10 induction

(A) Hoechst-stained nuclei and (B) anti-Hsp10/AF633 immunostaining of Hsp10 are imaged on the INCA6000 automated laser-based imaging platform with confocal modality at 40X objective magnification. (C) The two fused images allow to quantify by (D) automated image analysis the sum of Hsp10 immunostaining intensity per cell for the four imaged fields per well. Under our experimental conditions, Hsp10 localization is predominantly nuclear.

Figure 4. Cellular proliferation assessment in the presence or absence of seven transfection reagents

(A) Bar graph summary of the quantification of cellular proliferationbased on whole well imaging of Hoechst-stained nuclei and automated image analysis. Seven commercially available chemical transfection reagents are tested at 3 dilutions D1, D2 and D3 corresponding respectively to 0.025, 0.05 and 0.1 μL per well compared to control wells in absence of transfection reagent. Cell proliferation is measured at 24, 48 and 72 hours post transfectionwith chemical transfection reagent only and in combination with the EGFP DNA plasmid or scrambled siRNA duplex. (B) Bar graph summary of the quantification of TE based on whole well imaging and automated image analysis of Hoechst-stained nuclei and EGFP expression. TE is calculated as the percentage of EGFP-expressing cells as determined by the total count of EGFP masks overlapping with a Hoechst-stained nucleus mask compared to the total count of Hoechst-stained nuclei masks. Seven commercially available chemical transfection reagents are tested at 3 dilutions D1, D2 and D3 corresponding respectively to 0.025, 0.05 and 0.1 μL per well compared to control wells in absence of transfection reagent. TE is measured at 24, 48 and 72 hours post transfection with chemical transfection reagent only and in combination with the EGFP DNA plasmid or scrambled siRNA duplex.

Figure 4. Cellular proliferation assessment in the presence or absence of seven transfection reagents

(A) Bar graph summary of the quantification of cellular proliferationbased on whole well imaging of Hoechst-stained nuclei and automated image analysis. Seven commercially available chemical transfection reagents are tested at 3 dilutions D1, D2 and D3 corresponding respectively to 0.025, 0.05 and 0.1 μL per well compared to control wells in absence of transfection reagent. Cell proliferation is measured at 24, 48 and 72 hours post transfectionwith chemical transfection reagent only and in combination with the EGFP DNA plasmid or scrambled siRNA duplex. (B) Bar graph summary of the quantification of TE based on whole well imaging and automated image analysis of Hoechst-stained nuclei and EGFP expression. TE is calculated as the percentage of EGFP-expressing cells as determined by the total count of EGFP masks overlapping with a Hoechst-stained nucleus mask compared to the total count of Hoechst-stained nuclei masks. Seven commercially available chemical transfection reagents are tested at 3 dilutions D1, D2 and D3 corresponding respectively to 0.025, 0.05 and 0.1 μL per well compared to control wells in absence of transfection reagent. TE is measured at 24, 48 and 72 hours post transfection with chemical transfection reagent only and in combination with the EGFP DNA plasmid or scrambled siRNA duplex.

Figure 5. Whole well TE assessment of five transfection reagents

Fused whole well images of the DAPI and FITC channels captured at 4X objective magnification on the INCA2000 72 hours post transfection and showing the ensemble of Hoechst-stained nuclei and EGFP expressing cells. Representative images are shown for wells treated with 0, 0.025, 0.05 and 0.1 μL transfection reagent only or in combination with the EGFP DNA plasmid, for (A) DharmaFECT 1, (B) RNAiMAX, (C) FuGENE HD, (D) FuGENE 6 and (E) X-treme GENE 9.

Figure 5. Whole well TE assessment of five transfection reagents

Fused whole well images of the DAPI and FITC channels captured at 4X objective magnification on the INCA2000 72 hours post transfection and showing the ensemble of Hoechst-stained nuclei and EGFP expressing cells. Representative images are shown for wells treated with 0, 0.025, 0.05 and 0.1 μL transfection reagent only or in combination with the EGFP DNA plasmid, for (A) DharmaFECT 1, (B) RNAiMAX, (C) FuGENE HD, (D) FuGENE 6 and (E) X-treme GENE 9.

Figure 5. Whole well TE assessment of five transfection reagents

Fused whole well images of the DAPI and FITC channels captured at 4X objective magnification on the INCA2000 72 hours post transfection and showing the ensemble of Hoechst-stained nuclei and EGFP expressing cells. Representative images are shown for wells treated with 0, 0.025, 0.05 and 0.1 μL transfection reagent only or in combination with the EGFP DNA plasmid, for (A) DharmaFECT 1, (B) RNAiMAX, (C) FuGENE HD, (D) FuGENE 6 and (E) X-treme GENE 9.

Figure 6. Hsp10 induction assessment in the presence or absence of seven transfection reagents

Bar graph summary of the quantification of Hsp10 induction based on imaging and automated image analysis of Hoechst-stained nuclei and Hsp10 immunostaining as a function of time. Seven commercially available transfection reagents are tested at 3 dilutions D1, D2 and D3 corresponding respectively to 0.025, 0.05 and 0.1 μL per well compared to control wells in absence of transfection reagent. Hsp10 induction is measured at 24, 48 and 72 hours post transfection with chemical transfection reagent only and in combination with the EGFP DNA plasmid or scrambled siRNA duplex. Images on the Cy5 channel imaged at 40X objective magnification on the INCA6000 automated laser-based imaging platform with confocal modality showing Hsp10 expression.

Figure 7. Hsp70 induction assessment in the presence or absence of seven transfection reagents

Bar graph summary of the quantification of Hsp70 induction based on imaging and automated image analysis of Hoechst-stained nuclei and Hsp70 immunostaining as a function of time. Seven commercially available chemical transfection reagents are tested at 3 dilutions D1, D2 and D3 corresponding respectively to 0.025, 0.05 and 0.1 μL per well compared to control wells in absence of transfection reagent. Hsp70 induction is measured at 24, 48 and 72 hours post transfection with chemical transfection reagent only and in combination with the EGFP DNA or scrambled siRNA duplex. Images on the Cy5 channel imaged at 40X objective magnification on the INCA6000 automated laser-based imaging platform with confocal modality showing Hsp70 expression.