In vitro Infection of Neutrophils With Leishmania donovani Promastigotes

In vitro infection of neutrophils with L. donovani promastigotes

1. Adjust primary human neutrophil cell suspension to a concentration of 10 x 10⁶ cells/ml in complete medium 

2. Adjust L. donovani promastigote suspension (see culture conditions at the end of this protocol) to a concentration of 100 x 10⁶ cells/ml in complete medium

2. Add 1 ml of neutrophil suspension into wells of 12-well tissue culture plate. The number of wells depends on how many cells of the infected culture are needed for the subsequent work. Please note that during the washing steps (steps 5-7 in this protocol) about 50 % of the cells may be lost

3. Add 1 ml of L. donovani promastigote suspension and mix the cells with a 1 ml pipette

4. Incubate the plate for 3h at 37°C in a humidified air atmosphere containing 5% CO₂

5. Transfer the neutrophil-Leishmania co-cultures into conical 50 ml tube and fill the tube with PBS

6. Centrifuge the tube at 138 x g for 10 min at room temperature

7. Discard carefully the supernatant and resuspend the pellet with 5 ml PBS

8. Fill the tube with PBS and repeat the wash steps (steps 6-7 in this protocol) five more times

9. Discard the supernatant completely and resuspend the cells in 3 ml complete medium or in medium which is used in subsequent work 

10. Count cells and calculate cell concentration

11. Prepare cytocentrifuge slides from a small aliquote of the infected neutrophil suspension

12. Stain the slides with Giemsa and determine infection rate by visual assessment of intracellular Leishmania by using a light microscope with oil immersion objective and magnification of 1000 x

Complete medium:

RPMI 1640 medium (Sigma Aldrich) supplemented with 2 mM L-Glutamine (Merck, Darmstadt, Germany), 10 mM HEPES (Life Technologies, Darmstadt, Germany), 10 % heat-inactivated fetal calf serum (FCS, Gibco, Germany),100 U/mL Penicillin and 100 µg/mL Streptomycin (Biochrom, Berlin, Germany).

Leishmania donovani culture:

L. donovani promastigotes (strain MHOM/IN/82/Patna 1) are cultivated in Schneider´s Drosophila Medium w/L-Glutamine (Genaxxon, Ulm, Germany) supplemented with 10% FCS, 100 U/ml Penicillin, 100 µg/mL Streptomycin and 2% sterile filtered human urine at 27°C in a humidified air atmosphere containing 5% CO₂. For seeding of the culture a parasite density of 3 x 10⁶ cells/ml is used. The parasite counting is conducted in a hemocytometer with a chamber depth of 0.02 mm (0.0025 mm², depth 0.02 mm). Serial passaging is conducted until the passage 10.