A rapid method to assess the stage differentiation in Leishmania donovani by flow cytometry

Abstract

In this study we describe a rapid and novel method to assess the morphological stage differentiation in Leishmania donovani by flow cytometry (FCM). FCM is fast, accurate, and inexpensive to study the stage differentiation of promastigote into L. donovani axenic amastigote (LdAxAm). The non-flourimetric FCM method is easy to perform; with requirement of little expertise, and provides unambiguous results. It is an advanced tool, requires minimal time, and no fluorescent dyes. The gradual increase of differentiation and reduction in size from promastigote stage to LdAxAm leads to peak shifting from right to left on histogram. Earlier reports assessed the stage differentiation of Leishmania by studying the expression of stage specific markers like surface or secretory proteins and genes. For validation, conventional methods like microscopic analysis are used. These methods are quite expensive, laborious and time consuming. Non-flourimetric morphological parameters were further validated by conventional methods like optical and scanning electron microscopy. Additionally, differential expression of stage specific genes (e.g. upregulation of amastin and ATP binding cassette A3 (ABCA3) transporter gene transcripts) and differential activity of enzymes (down regulation of secretory acid phosphatase (SAcP) and 3'-nucleotidase enzyme activity) in LdAxAm suggest stage differentiation. Therefore, we believe that our method is an alternative tool for high reproducibility and reliability in assessment of stage differentiation.