Preparation of effector cells

1 Plate macrophages in Lab Tek tissue culture slides at 1 x 105 cells/well in 0.5 ml complete RPMI. U937 and THP-1 are also differentiated in Lab Tek tissue culture slides at 5 x 104 cells/well in 0.5 ml RPMI complete medium containing PMA (10 jig/ml) or retinoic acid (RA) (10~6 M).

2 Incubate at 37°C to allow the cells to adhere (3-4 h for macrophages, three days for differentiating U937 or THP-1).

3 Remove non-adherent cells by three extensive washings with warm complete RPMI.

4 Add the appropriate macrophage activators in 0.1 ml of medium to a final volume of 0.5 ml.

5 Incubate the cells for 18-24 h at 37°C.

6 Remove supernatant by aspiration.

7 Wash the cells three times with warm complete RPMI.

C Assay

1 Add 0.5 ml of parasite suspension (1-2 x 106 parasites/ml to have a 10:1 parasite-to-host ratio) to Lab Tek tissue culture slides containing washed monocyte/macrophage monolayers.

2 Incubate for4 h at 37°C.

3 Remove non-internalized parasites by three washings with warm RPMI complete medium (eliminate recovered parasites using an appropriate biohazard disposal).

4 Culture the infected cells in medium, replacing eveiy 24 h.

5 Fix and stain the slides with Giemsa Plus reagent at the desired times.

6 Observe the slides at x 1000 with an immersion objective.

7 Count at least 200 macrophages chosen at random in non-contiguous fields. Determine microbicidal activity, defined as decrease in infected macrophages in treated cultures relative to control cultures by the following formula:

{(% infected control macrophages - % infected treated macrophages) / (% infected control macrophages)} x 100.

Results are expressed as per cent LiisfirmiJita-infected macrophages ± SEM standard error of the mean of replicate samples.

3The cell lines (U937, THP-t) need to be differentiated into non-dividing, plastic adherent monolayers utilizing PMA (10 ng/ml) (Sigma Chemical) or retinoic acid (RA 10"fi M) (BIOMOL Research Lab.), respectively, for three days (44).

b A long-term maintenance in vitro causes a decrease in virulence. To maintain the virulence, a monthly passage in vivo is necessary. Promastigotes are very mobile and can be counted more easily in a haemocytometer after fixation with 2% formaldehyde. The infectivity of Leishmania promastigotes varies according to the growth phase: during the logarithmic growth phase the cells are actively dividing and less infectious than in the stationary growth phase.

c The concentration should be adjusted to achieve the desired para site-to-host ratio.

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