fluorescent dna binding dye celltox green  (Promega)

Journal: mBio

Article Title: The Bordetella effector protein BteA induces host cell death by disruption of calcium homeostasis

doi: 10.1128/mbio.01925-24

Figure Lengend Snippet: BteA-induced cell death is characterized by fragmentation of the endoplasmatic reticulum and mitochondrial networks. ( A ) Time lapse analysis of morphological changes and plasma membrane permeabilization in Hela cells. Hela cells were infected with B. bronchiseptica wild-type ( Bb WT) and Bb Δ bteA mutant ( Bb Δ bteA ), expressing the fluorescent protein mNeonGreen, at MOI of 10:1 in the presence of propidium iodide (5 µg/mL). A sequence of time lapse images is shown. Bright field, gray; bacteria, cyan; propidium iodide, magenta. Scale bar, 20 µm. Data are representative of three independent experiments. ( B and C ) Comparison of B. bronchiseptica and B. pertussis cytotoxicity against HeLa cells. HeLa cells were infected with B. bronchiseptica ( B ) or B. pertussis ( C ) wild-type strains and their mutant derivatives at the indicated MOI. Plasma membrane permeabilization was determined using the fluorescent DNA binding dye CellTox Green. For B. bronchiseptica , infections were conducted both in the presence (gly+) and absence of glycine (5 mM). Asterisks indicate a statistically significant difference ( P < 0.05, unpaired two-tailed t -test) between Bb WT and Bb WT gly+ infection samples at 4 h. Data represent the mean ± SEM of a representative experiment out of 2 performed in technical triplicate. ( D ) Visualization of cellular structures. Hela cells were transfected to express fluorescent proteins tagged with localization signals for endoplasmatic reticulum (ER) and mitochondria (mito). One hour after infection with Bb WT and Bb Δ bteA mutant at MOI 50:1 , cells were fixed and analyzed by fluorescence imaging. ER, yellow; mitrochondria, magenta. Scale bar, 20 µm. The shown micrographs are representative of two independent experiments from which the organelle morphology was scored. Analysis was performed on at least 100 cells per experiment and condition and is plotted as morphology % ± SEM. Asterisks indicate statistically significant differences ( P < 0.05, unpaired two-tailed t -test) between the % of fragmented ER or mito compared with uninfected cells.

Article Snippet: Cytotoxicity of B. bronchiseptica and B. pertussis toward HeLa and HEK-Cas9 was determined as changes in cell membrane integrity using the fluorescent DNA binding dye CellTox Green (Promega, Cat. No. G8743), as described previously ( ).

Techniques: Clinical Proteomics, Membrane, Infection, Mutagenesis, Expressing, Sequencing, Bacteria, Comparison, Binding Assay, CellTox Assay, Two Tailed Test, Transfection, Fluorescence, Imaging

Journal: mBio

Article Title: The Bordetella effector protein BteA induces host cell death by disruption of calcium homeostasis

doi: 10.1128/mbio.01925-24

Figure Lengend Snippet: BteA disrupts cytosolic calcium homeostasis, leading to the permeabilization of cell plasma membrane. ( A and B ) Calcium influx precedes B. bronchiseptica -induced plasma membrane permeabilization. HeLa cells were infected with Bb WT and Bb Δ bteA mutant at the indicated MOI. Calcium influx was monitored using cytosolic Ca 2+ indicator Fluo-4/AM ( A ), whereas plasma membrane permeabilization was determined in parallel wells by fluorescent DNA binding dye CellTox Green ( B ). Data represent the mean ± SEM of a representative experiment out of 3 performed in technical duplicate. ( C ) Calcium imaging. HeLa cells loaded with cytosolic Ca 2+ indicator Fluo-4/AM were infected with Bb WT and Bb Δ bteA , expressing the fluorescent protein mScarlet (mSc), at MOI of 10:1. Sequence of time lapse images is shown. Bacteria, magenta; cytosolic Ca 2+ indicator Fluo-4/AM, yellow. Scale bar, 20 µm. Data are representative of two independent experiments. ( D and E ) Treatment with 2-APB delays calcium influx and host cell death. HeLa cells were pre-incubated with 100 µM 2-APB (2-APB+) for 30 min, or left untreated, before being infected with Bb WT or Bb Δ bteA derivative at MOI 25:1. Calcium influx was assessed using Fluo-4/AM Ca 2+ indicator, ( D ) whereas plasma membrane permeabilization was determined in parallel wells by fluorescent DNA binding dye CellTox Green ( E ). Asterisks indicate a statistically significant difference ( P < 0.05, unpaired two-tailed t -test) between Bb WT and Bb WT 2-APB+ infection samples at 1 h 30 min (maximum of Bb WT Fluo-4/AM signal) ( D ) and at 4 h ( E ). Data represent the mean ± SEM of a representative experiment out of three performed in technical duplicate.

Article Snippet: Cytotoxicity of B. bronchiseptica and B. pertussis toward HeLa and HEK-Cas9 was determined as changes in cell membrane integrity using the fluorescent DNA binding dye CellTox Green (Promega, Cat. No. G8743), as described previously ( ).

Techniques: Clinical Proteomics, Membrane, Infection, Mutagenesis, Binding Assay, CellTox Assay, Imaging, Expressing, Sequencing, Bacteria, Incubation, Two Tailed Test