chicken embryonic fibro blast cell line df1  (ATCC)

Journal: bioRxiv

Article Title: Influenza A virus infection alters lipid packing and surface electrostatic potential of the host plasma membrane

doi: 10.1101/2023.07.25.550511

Figure Lengend Snippet: HEK293T and DF1 cells were either: non-infected (MOCK), treated with DOPS-SUV (DOPS, positive control), infected with FPV or with WSN influenza A strains. All cells were expressing the FRET-sensor MCS+ and emission spectrum images (22 spectral channels from 499 nm to 695 nm) were acquired 16 hpi using 488 nm excitation. (A) Average normalized emission spectra of all the selected regions of interest (ROI) at the equatorial plane of HEK293T and DF1 cells expressing MCS+, following the indicated treatment. Data are represented as mean ± SD from 50-55 HEK293T cells and 21-33 DF1 cells. (B) Representative ratiometric FRET images (RG ratio, pseudo-colored as indicated by the color scale) of HEK293T and DF1 cells expressing MCS+. White rectangles represent examples of ROIs at the PM selected for FRET quantification. Scale bars represent 10 µm. (C) RG ratio derived from the average intensity spectra of each cell type for the indicated treatment. Data from two separate experiments were pooled, plotted and analyzed using one-way ANOVA Tukey’ ss multiple comparison test (**** p < 0.0001). Each data point represents the average value measured for a ROI at the PM in one cell (Table S2).

Article Snippet: Madin-Darby canine kidney type II (MDCK II) cells (ECACC 00062107, European Collection of Authenticated Cell Cultures, Porton Down, UK), chicken embryonic fibro-blast cell line DF1 (ATCC number: CRL–12203, kindly provided by Andreas Herrmann, Humboldt University Berlin, Germany) and human embryonic kidney (HEK) cells from the 293T line (CRL-3216TM, purchased from ATCC, Kielpin Lomianki, Poland) were maintained in phenol red-free, high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum in a humidified incubator at 37°C and 5% CO 2 atmosphere.

Techniques: Infection, Positive Control, Expressing, Derivative Assay, Comparison

Journal: bioRxiv

Article Title: Influenza A virus infection alters lipid packing and surface electrostatic potential of the host plasma membrane

doi: 10.1101/2023.07.25.550511

Figure Lengend Snippet: HEK293T and DF1 cells were either: non-infected (MOCK), treated with methyl-β-cyclodextrin (MbCD), infected with FPV or with WSN influenza A strains. All cells were labelled with the solvatochromic probes Laurdan (A-C) and Di-4-ANEPPDHQ (D-F) , and then imaged 16 hpi. Averaged normalized fluorescence emission spectra of all selected regions of interest (ROI) at the equatorial plane of HEK293T and DF1 cells stained with Laurdan (A) or Di-4-ANEPPDHQ (D) , for the indicated treatment. Data are represented as mean ± SD of 52-110 cells stained with Laurdan and 36-127 cells stained with Di-4-ANEPPDHQ (Table S3 and S4). Representative ratiometric GP images (GP index, pseudo-colored as indicated by the color scale) of HEK293T and DF1 cells stained with Laurdan (B) or Di-4-ANEPPDHQ (E) . White lines represent examples of ROIs at the PM selected for GP index quantification. Scale bars represent 10 µm. GP index derived from the average intensity spectra from Laurdan- (C) or Di-4-ANEPPDHQ-stained (F) cells for each cell type and indicated treatment. Data from three separate experiments were pooled, plotted and analyzed using one-way ANOVA Tukey’ ss multiple comparison test (**** p < 0.0001). Each data point represents the average value measured for a ROI at the PM in one cell (Tables S3 and S4).

Article Snippet: Madin-Darby canine kidney type II (MDCK II) cells (ECACC 00062107, European Collection of Authenticated Cell Cultures, Porton Down, UK), chicken embryonic fibro-blast cell line DF1 (ATCC number: CRL–12203, kindly provided by Andreas Herrmann, Humboldt University Berlin, Germany) and human embryonic kidney (HEK) cells from the 293T line (CRL-3216TM, purchased from ATCC, Kielpin Lomianki, Poland) were maintained in phenol red-free, high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum in a humidified incubator at 37°C and 5% CO 2 atmosphere.

Techniques: Infection, Fluorescence, Staining, Derivative Assay, Comparison

Journal: bioRxiv

Article Title: Influenza A virus infection alters lipid packing and surface electrostatic potential of the host plasma membrane

doi: 10.1101/2023.07.25.550511

Figure Lengend Snippet: Quantitative analysis of protein diffusion via fluorescence correlation spectroscopy (sFCS) in non-infected (MOCK) and FPV-/WSN-infected HEK293T and DF1 cells expressing three model proteins labelled with green fluorescent proteins (mEGFPs) and associated to the plasma membrane (PM). Specifically, we investigated: i) a construct anchored to the inner-leaflet of the PM via a myristoylated and palmitoylated (mp) peptide (mp-mEGFP), ii) a construct anchored to the outer-leaflet of the PM via a glycosylphosphatidylinositol (GPI) anchor (GPI-mEGFP) and iii) one representative transmembrane protein, i.e. the influenza envelope protein hemagglutinin (HA-mEGFP). Measurements were performed 16 hpi. The box plots show the diffusion coefficients calculated from sFCS diffusion times. Data from three separate experiments were plotted and analyzed using one-way ANOVA Tukey’ ss multiple comparison test (* p < 0.05, *** p < 0.001, **** p < 0.0001). Each data point represents the value measured at the PM in one cell (Table S5).

Article Snippet: Madin-Darby canine kidney type II (MDCK II) cells (ECACC 00062107, European Collection of Authenticated Cell Cultures, Porton Down, UK), chicken embryonic fibro-blast cell line DF1 (ATCC number: CRL–12203, kindly provided by Andreas Herrmann, Humboldt University Berlin, Germany) and human embryonic kidney (HEK) cells from the 293T line (CRL-3216TM, purchased from ATCC, Kielpin Lomianki, Poland) were maintained in phenol red-free, high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum in a humidified incubator at 37°C and 5% CO 2 atmosphere.

Techniques: Diffusion-based Assay, Fluorescence, Spectroscopy, Infection, Expressing, Clinical Proteomics, Membrane, Construct, Comparison