Flow Cytometric Assessment of Drug Susceptibility in Leishmania infantum Promastigotes

Rafael Nuñez1, Sarah Kamau1, Felix Grimm1

1 University of Zürich, Zürich, Switzerland
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.14
DOI:  10.1002/0471142956.cy1114s15
Online Posting Date:  May, 2001
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Abstract

Flow cytometry can be a valuable tool for parasitology in studies of parasite‐drug interaction and cellular toxicity. This unit presents protocols for assessment of Leishmania promastigote proliferation, viability, and cellular protein content. The cytometric approach permits one to detect, differentiate, and quantify cellular changes in these parasites resulting from drug treatment, in this case allopurinol, as well as demonstrate differences in drug susceptibility between promastigote forms. Rapid and relatively simple flow cytometry replaces incorporation of [3H]‐thymidine, hitherto the most common method for determining cell division.

     
 
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Table of Contents

  • Basic Protocol 1: Assessing Leishmania Promastigote Proliferation by Staining with CFSE
  • Basic Protocol 2: Assessing Leishmania Promastigote Viability by Staining with PI and SYBR‐14
  • Basic Protocol 3: Assessing Cellular Protein Content of Promastigotes by Staining with FITC
  • Support Protocol 1: Generation of Allopurinol‐Resistant Promastigotes (allo‐p229)
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Assessing Leishmania Promastigote Proliferation by Staining with CFSE

  Materials
  • Promastigote forms of L. infantum (MCAN/ES/89/IPZ229/I/89, zymodeme MON 1: wt‐p229) isolated from a patient (see recipe for cultivation)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 2.8 mg/ml 5‐(and‐6)‐carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes) in DMSO (store up to 1 year at −20°C)
  • Medium for Leishmania promastigote culture (see recipe)
  • 10 mg/ml allopurinol stock in 0.1 N NaOH (store up to 2 to 4 weeks at 4°C)
  • 15‐ml conical centrifuge tubes (e.g., Corning)
  • 25‐cm2 tissue culture flasks (Corning)
  • 27°C incubator
  • 5‐ml round‐bottom polystyrene centrifuge tubes (e.g., Falcon)
  • Flow cytometer (e.g., FACScalibur; Becton Dickinson)
  • Additional reagents and equipment for counting cells in a hemacytometer ( appendix 3A)

Basic Protocol 2: Assessing Leishmania Promastigote Viability by Staining with PI and SYBR‐14

  Materials
  • Promastigote forms of L. infantum (MCAN/ES/89/IPZ229/I/89, zymodeme MON 1: wt‐p229) isolated from a patient (see recipe for cultivation)
  • Medium for Leishmania promastigote culture (see recipe)
  • 10 mg/ml allopurinol stock in 0.1 N NaOH (store up to 4 weeks at 4°C)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1 mg/ml propidium iodide (PI) stock in water (store up to 1 year at −20°C)
  • 1 mg/ml SYBR‐14 (Molecular Probes) in DMSO (store up to 1 year at −20°C protected from light)
  • 25‐cm2 tissue‐culture flasks (Corning)
  • 15‐ml conical centrifuge tubes (e.g., Corning)
  • 27°C incubator
  • 5‐ml round‐bottom polystyrene centrifuge tubes (e.g., Falcon)
  • Flow cytometer (e.g., FACScalibur; Becton Dickinson)
  • Additional reagents and equipment for counting cells in a hemacytometer ( appendix 3A)

Basic Protocol 3: Assessing Cellular Protein Content of Promastigotes by Staining with FITC

  Materials
  • Promastigote forms of L. infantum (MCAN/ES/89/IPZ229/I/89, zymodeme MON 1: wt‐p229) isolated from a patient (see recipe for cultivation)
  • Medium for Leishmania promastigote culture (see recipe)
  • 10 mg/ml allopurinol in 0.1 N NaOH (store up to 2 to 4 weeks at 4°C)
  • PBS‐DNase: 4 µg/ml DNase (Type I; Sigma) in PBS ( appendix 2A); store up to 1 year at −20°C
  • 70% methanol
  • 1 mg/ml fluorescein isothiocyanate (FITC; Molecular Probes) in PBS (prepare fresh)
  • 25‐cm2 tissue‐culture flasks (Corning)
  • 27°C incubator
  • 15‐ml conical centrifuge tubes (e.g., Corning)
  • 5‐ml round‐bottom polystyrene centrifuge tubes (e.g., Falcon)
  • Flow cytometer (e.g., FACScalibur; Becton Dickinson)
  • Additional reagents and equipment for counting cells in a hemacytometer ( appendix 3A)

Support Protocol 1: Generation of Allopurinol‐Resistant Promastigotes (allo‐p229)

  Materials
  • Promastigote forms of L. infantum (MCAN/ES/89/IPZ229/I/89, zymodeme MON 1: wt‐p229) isolated from a patient (see recipe for cultivation)
  • Medium for Leishmania promastigote culture (see recipe)
  • 10 mg/ml allopurinol stock in 0.1 N NaOH (store up to 2 to 4 weeks at 4°C)
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Figures

Videos

Literature Cited

Literature Cited
   Baneth, G., Dank, G., Keren‐Kornblatt, E., Sekeles, E., Adini, I., Eisenberger, C.L., Schnur, L.F., King, R., and Jaffe, C.L. 1998. Emergence of visceral leishmaniasis in central Israel. Am. J. Trop. Med. Hyg. 59:722‐725.
   Brun, R. and Schönenberger, M. 1979. Cultivation and in vitro cloning of procyclic culture forms of Trypanosoma brucei in a semi‐defined medium. Acta Tropica 36:289‐292.
   Cunningham, I. 1977. New culture medium for maintenance of tsetse tissues and growth of trypanosomatids. J. Protozool. 24:325‐329.
   Frayha, G.J., Smyth, J.D., Gobert, J.G., and Savel, J. 1997. The mechanism of action of antiprotozoal and anthelmintic drugs in man. Gen. Pharmacol. 28:273‐299.
   Garner, D.L., Johnson, L.A., Yue, S.T., Roth, B.L., and Haugland, R.P. 1994. Dual DNA staining assessment of bovine sperm viability using SYBR‐14 and propidium iodide. J. Androl. 15:620‐629.
   Grimm, F., Brun, R., and Jenni, L. 1991. Promastigote infectivity in Leishmania infantum. Parasitol. Res. 77:185‐191.
   Kamau, S., Hurtado, M., Müller‐Doblies, U., Grimm, F., and Nunez, R. 2000. Flow cytometric assessment of allopurinol susceptibility in Leishmania infantum promastigotes. Cytometry 40:353‐360.
   Montalban, C., Calleja, J.L., Erice, A., Laguna, F., Clotet, B., Podzamczer, D., Cobo, J., Mallolas, J., and Yebra, M., Gallego, M. 1990. The Co‐operative Group for Study of Leishmaniasis in AIDS: Visceral leishmaniasis in patients infected with human immunodeficiency virus. J. Infect. 21:261‐270.
   Quellette, M. and Papadopoulou, B. 1993. Mechanisms of drug resistance in Leishmania. Parasitol. Today 9:150‐153.
Key Reference
   Kamau et al., 2000. See above.
  The study of Kamau et al. demonstrated (1) that CFSE determined the effect of the antileishmania drug allopurinol on the proliferation of promastigotes at different time intervals, (2) that SYBR‐14 used in combination with PI is effective for simultaneously visualizing both the living and dead populations of Leishmania promastigotes before and after treatment with allopurinol, and (3) that the total cellular protein content of fixed promastigotes can also be determined by staining with FITC.
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