imaging flow cytometry analysis  (DiaSorin Biotechnology)

Journal: iScience

Article Title: The canonical single-stranded DNA-binding protein is not an essential replication factor but an RNA chaperon in Saccharolobus islandicus

doi: 10.1016/j.isci.2023.108389

Figure Lengend Snippet: Growth and flow cytometry analysis of the knockout strains of Sisssb and Sisdbp (A) PCR verification of the knockout strains Δ ssb , Δ dbp , and Δ dbp Δ ssb . Genomic DNA and two primer pairs, flanking F/R and gene specific F/R, were used for the analysis. (B) Western blotting analysis of the knockout strains using whole-cell lysate. Cells were collected at OD 600 = 0.5–0.8, disrupted by sonication. Primary antibody of SisDBP was added alone, while those of SisTBP and SisSSB were incubated simultaneously. Anti-SisTBP was used as the loading control. (C) Growth curves under normal conditions. Cells were cultured with shaking at 110 rpm and 75°C in STVU medium with an initial OD 600 0.05. The values were calculated based on three biological repeats. (D) Flow cytometry analysis of the knockout strains. Acetic acid (6 mM）was added into the culture medium when the OD 600 reached 0.15–0.2. Samples were taken at different time (0, 1, 2, 3, 4, 5, 6, 7, and 8 h) and analyzed as described in the materials and methods. “1C” and “2C” indicate one and two copies of chromosomes. (E) Growth curves of cells treated with 4-NQO. Cells were cultured as in (C) except that 4-NQO (3 μM）was added into the culture medium when the OD 600 reached 0.2 (at 12 h). The values were calculated based on three biological repeats. See also Figure S2 .

Article Snippet: The DNA content of the cells was analyzed using the ImageStreamX MarkII Quantitative imaging analysis for flow cytometry system (Merck Millipore, Germany).

Techniques: Flow Cytometry, Knock-Out, Western Blot, Sonication, Incubation, Control, Cell Culture

Journal: iScience

Article Title: The canonical single-stranded DNA-binding protein is not an essential replication factor but an RNA chaperon in Saccharolobus islandicus

doi: 10.1016/j.isci.2023.108389

Figure Lengend Snippet: SSB-deficient cells exhibited slow growth at lower temperature (A) Growth curves of SSB-deficient strains at 55°C. Cells were cultured in liquid STVU at 75°C to OD 600 = 0.6–0.8 and then used as inoculates with an initial OD 600 of 0.05. The cultivation was continued in STVU medium or in the arabinose-containing medium ATVU (indicated with “A”) at 55°C with shaking. The optical density was monitored and the values were calculated based on measurements of three biological replicates. (B) Cytometry profiles of the strains grown at 55°C. Samples were taken at 72 h and analyzed. Cells of E233S cultivated to middle logarithmic phase (OD 600 ∼0.4) at 75°C was used as a control. See also Figure S3 .

Article Snippet: The DNA content of the cells was analyzed using the ImageStreamX MarkII Quantitative imaging analysis for flow cytometry system (Merck Millipore, Germany).

Techniques: Cell Culture, Cytometry, Control

Journal: iScience

Article Title: The canonical single-stranded DNA-binding protein is not an essential replication factor but an RNA chaperon in Saccharolobus islandicus

doi: 10.1016/j.isci.2023.108389

Figure Lengend Snippet: Overexpression of SisSSB has global effect on cell cycle progression and growth (A) Assay of the expression levels in the promoter replacement strain Para::ssb under non-inducible and inducible conditions by western blotting. Cells were cultured in liquid STVU or ATVU medium and collected at OD 600 0.5–0.8. The cells were then disrupted by sonication and the cell lysates were subjected to SDS-PAGE and western blotting analysis with antibodies against SisSSB and SisTBP at the same time. (B) Quantitative analysis of the results in (A). The values were calculated based on three replicates. Error bars indicated the standard deviation. (C) Growth curves of the promoter substitution strain in comparison with the wild-type E233S. The cells were cultured in liquid ATVU medium with an initial OD 600 0.05 with shaking (110 rpm) at 75°C. OD 600 was monitored at 6 or 12 h interval. The values were based on measurements of three biological replicates. (D) Comparison of the cytometry profiles of the synchronized E233S and the promoter substitution stain. The cells were cultured in TSVU medium until the OD 600 reached 0.15–0.2 when acetic acid (6 mM) was added into the culture. After cultured for 3 h, 0.2% D-arabinose was added to induce the expression of SisSSB. After incubation for further 6 h, samples were taken at different time points (0, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, 10, 11, and 12 h) and subjected to the flow cytometry analysis. “1C” and “2C” indicate the cells containing one and two copies of chromosomes, respectively. (E) Comparison of the cell cycle periods of E233S and the SisSSB overexpression strain according to (D). G2→M→G1 (green), from the cell cycle release to 1C appearance; G1→S→G2 (gray): 1C appearance to 2C peak. See also Figure S1 .

Article Snippet: The DNA content of the cells was analyzed using the ImageStreamX MarkII Quantitative imaging analysis for flow cytometry system (Merck Millipore, Germany).

Techniques: Over Expression, Expressing, Western Blot, Cell Culture, Sonication, SDS Page, Standard Deviation, Comparison, Cytometry, Staining, Incubation, Flow Cytometry