Journal: bioRxiv

Article Title: Distinct features of the Leishmania cap-binding protein LeishIF4E2 revealed by CRISPR-Cas9 mediated heterozygous deletion

doi: 10.1101/2020.05.13.093914

Figure Lengend Snippet: The LeishIF4E2(+/-) deletion mutant shows altered promastigote morphology. (A) Mid-log phase (Day 2) promastigotes of wild type, Cas9/T7 expressers, LeishIF4E2(+/-) cells, and LeishIF4E2 add-back cells were fixed with 2% paraformaldehyde and visualized by phase contrast microscopy at 100x magnification. Wild type (WT), Cas9/T7 expressing cells showed normal promastigote morphology while LeishIF4E2(+/-) became round with reduced flagellar length. This altered phenotype was reverted back to normal in LeishIF4E2 add-back cells. (B) All the cell lines were stained with 20 µg/mL propidium iodide (PI) for 30 minutes and cell viability was analyzed using the ImageStream X Mark II Imaging flow cytometer (Millipore). 20,000 cells were analyzed for each sample and percent viable cells were determined. (C) .The circularity of single, viable and focused cells from each of the cell lines was quantified using flow cytometry and shown as percentage of the total number of cells measured. Data from three independent experiments are shown.

Article Snippet: Flow cytometry analysis of LeishmaniaCell viability was verified by incubation of the cells with 20 µg/mL propidium iodide (PI) during 30 minutes.

Techniques: Mutagenesis, Microscopy, Expressing, Staining, Imaging, Flow Cytometry