L. donovani MHOM/SD/00/1S (24 (
link)) promastigotes were grown in medium
M-199,
Earle’s salts (Biological Industries Ltd.) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco Ltd.) and a 1%
penicillin-streptomycin solution (Biological Industries Ltd.). Axenic differentiation of
L. donovani promastigotes to amastigotes was carried out as described previously (7 (
link)). Briefly, mid-log-phase promastigotes were washed twice in Earle’s salt solution and finally suspended in amastigote medium containing
M-199 at pH 5.5 supplemented with 0.5 mM sodium succinate, 25% bovine serum, and 1%
penicillin-streptomycin solution. Mature amastigotes developed at 5 days after exposure of promastigotes to the differentiation medium.
Arginine deprivation was carried out as described previously by Pawar et al. (12 (
link)). Briefly, mid-log-phase promastigotes (1 × 10
7 cells/ml) were washed with Earle’s balanced salt solution twice and resuspended in arginine-deficient medium
M-199 (Biological Industries Ltd.) at 26°C for the specified period before being transferred to ice. Arginine-deprived cells were washed twice with ice-cold Earle’s balanced salt solution before being used for transport assays and Northern and Western blot analyses.
