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Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites
Published on: March 16, 2018
Separation of Leishmania-infected macrophages by step-SPLITT fractionation
Mauricio Hoyos1, Andrea Niño, Manuel Camargo
1Laboratoire de Physique et Mécanique des Milieux Hétérogènes, UMR7636 CNRS, Ecole Supérieure de Physique et Chimie Industrielles, ESPCI, 10 rue Vauquelin, 75231 Paris Cedex 05, France. hoyos@pmmh.espci.fr
Abstract:
After a primary infection protocol of macrophages with Leishmania amazonensis, the percentage of infection drops as infection progresses and the uninfected population of macrophages mask the effects of infection for electrophysiological studies. In order to increase or maintain the infection percentage, we introduce an enrichment process after primary infection, which increases the possibility of following the infection longer times than any known process. A membraneless separation technique, step-SPLITT fractionation, implying flow and transverse gravity field in a ribbon-like channel, was used for enriching samples of macrophages infected with particles and with L. amazonensis. We demonstrate the capability of the s-SPLITT of generating, from a mixture resulting from a primary infection, an enriched and a depleted fraction with infected cells, without using any selective labeling pre-processing. It is also shown that a continuous sorting is possible without damaging cells and the losses of matter into the separation chamber is minimal.
Insights
This study introduces step-SPLITT fractionation to enrich Leishmania amazonensis-infected macrophages, enabling longer observation times. This method effectively separates infected from uninfected cells without labeling, improving infection studies.
Area of Science:
- Cell Biology
- Parasitology
- Biotechnology
Background:
- Macrophage infection by Leishmania amazonensis decreases over time, complicating electrophysiological studies.
- Uninfected macrophages can mask the effects of infection in experimental settings.
Purpose of the Study:
- To develop an enrichment method for Leishmania amazonensis-infected macrophages.
- To enable longer-term observation of infected macrophages by maintaining infection percentages.
Main Methods:
- Utilized step-SPLITT (Sustained Particle Loading and Immobilization Through Transport) fractionation, a membraneless separation technique.
- Applied a flow and transverse gravity field in a ribbon-like channel to separate infected and uninfected macrophages.
- Enriched macrophage samples infected with Leishmania amazonensis.
Main Results:
- Demonstrated the capability of step-SPLITT to generate enriched and depleted fractions of infected cells from a primary infection mixture.
- Achieved cell enrichment without the need for selective labeling pre-processing.
- Showcased continuous cell sorting without causing cell damage and with minimal material loss.
Conclusions:
- Step-SPLITT fractionation is an effective method for enriching Leishmania amazonensis-infected macrophages.
- This technique facilitates extended study of infected cells, overcoming limitations of natural infection decline.
- The method offers a label-free, continuous, and non-damaging approach for cell separation in parasitological research.
