For in vitro infection studies; J774A.1 macrophages were plated on coverslips at the density of 1 X105 cells/ coverslip in a 6-well culture plate or at a density of 5 X 105 cells/well of a 6-well culture plate, and allowed to rest for 12-18 hrs. The cells were challenged with stationary phase parasites at a multiplicity of infection (MOl) of 1:10 for 6 hrs after which the excess unbound parasites were washed with phosphate buffered saline or plain medium and the macrophages incubated at 37°C for different time points. Infection was visualised by staining the cells with cell permeant nucleic acid stain Syto 11 (Molecular Probes, Eugene, OR) in the dark for 10 min and viewed under a Nikon Eclipse TE2000E fluorescence microscope (Nikon, Japan)

In vivo infection of Syrian hamsters with Leishmania donovani parasites

In vitro infection of J774A.l murine macrophages with L. donovani parasites

0% FBS and cultured as described above. The cells usually regain motility by 24 to 48 hrs and the culture is ready for use after splitting them once

For long term storage, Leishmania donovani were stored as Dimethyl Sulfoxide (DMSO) containing frozen stocks (freeze downs). Parasites in mid to late log phase were taken and dead and agglutinated cells removed by centrifugation at 129 x g for 10 min. The supernatant was centrifuged at 1258 x g for 10 min to pellet cells which were then washed 2X with plain medium followed by resuspension in FBS containing 10% DMSO as the cryoprotectant. These were transferred to a labelled cryogenic vial and immediately transferred to -20°C for a few hours, followed by incubation at -70°C overnight. The following day, the stocks were transferred to liquid nitrogen (liq N2). To revive a frozen stock, the stock was retrieved from liq N2 and transferred to a beaker containing water at ~37°C. Once the stock has thawed, the cells were immediately transferred to lOmL plain mDMEM and centrifuged at 217 x g for 10 min. The pellet was washed three to four times with plain medium followed by resuspension in mDMEM containing

Preparation and revival of frozen stocks of Leishmania donovani

Murine macrophage cell line J774A.1 (ATCC no. TIB-67) was maintained in phenol red free DMEM supplemented with 10% heat inactivated (45 min at 65°C) foetal bovine serum at 37°C in 5% C02 and 95% air. The cultures were sub-cultured every three days or at the attainment of 80 % confluency.

In vitro J774A.l murine macrophage cultur

Long-term axenic amastigotes were generated by subjecting promastigotes to pH and temperature modulations as described elsewhere (Debrabant et al., 2004). Briefly, live metacyclic promastigotes were harvested by centrifugation and resuspended in DMEM containing 20% FBS and a pH of 5.5 and sub-cultured at 23°C after 72 h three times. Following this, the cells were then transferred to 37°C, 5% C02 for 3 passages after 72 h each. Axenic amastigotes obtained after the last subculture was stained with Giemsa stain and checked under the microscope. They were then maintained at 37°C in a humidified atmosphere containing 5% C02 in air

Leishmania donovani promastigotes (MHOM/IN/80/DD8) were obtained from Dr. R Vishwakarma from the National Institute of Immunology, New Delhi, India. These were grown routinely on blood agar slants containing 1% glucose, 5.2% brain heart infusion agar extract, 6%(v jv) of rabbit blood and 1mg/mL of gentamycin as antibiotic (Sudhandiran and Shaha, 2003) at 23°C. After three days of culture on slants, fresh slants were streaked using a loop for regular maintenance. For liquid cultures, cells were transferred from a slant to modified DMEM (3. 7 g Sodium bicarbonate, 5. 96g HE PES, 5mg Hemin, 1mg Biotin, 13.36mg Adenine, 7.6mg Xanthine, 0.5rnl Triethanolamine, 40mg Tween 80) with 10 % foetal bovine serum (FBS). Before experiments, the cells were centrifuged at 129 x g for 10 min to remove dead and agglutinated parasites; the supernatant was centrifuged at 1258 x g for 10 min to pellet the live cells which were then resuspended in appropriate amounts of media for experiments

In vitro Leishmania donovani culture

Cell Culture Techniques

METHODS

Ethylene glycol tetraacetic acids (EGTA), Ethylene diamine tetraacetic acid (EDTA), Dimethyl Sulfoxide (DMSO), Dimethyl formamide (DMF) and Triton-X 114 were obtained from Sigma Chemical Company (St. Louis, MO). Heptane, hexane, chloroform, methanol and ethyl acetate (HPLC grade) were obtained from Merck Specialities Pvt. Ltd. (Mumbai, India). All chemicals were of the highest reagent grade. Other chemicals, unless specified, were from Sigma Chemical Company (St. Louis, MO). All plastic-wares were purchased from Nunc (Denmark), BD Falcon (Franklin Lakes, NJ), Axygen (Union City, CA) or Griener Bio-One (Frickenhausen, Germany

Iris, Acrylamide, N, N'-methylene-bis-acrylamide, Ammonium persulphate (APS), N,N,N' ,N' ,-tetramethylene-diamine (TEMED), and Coomassie Blue R-250 were obtained from Sigma Chemical Company (St. Louis, MO). CB-XlM Protein Assay kit was purchased from G-Biosciences (Maryland Heights, MO). RainbowlM protein molecular weight markers were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). Amersham LMWlM Calibration kit for SDS electrophoresis, Amersham ECL™ DualValue Western Blotting Marker was obtained from GE Healthcare (Buckinghamshire, UK). Enhanced chemiluminescence (ECL) components (Hypercassette Blue, Hyper-film ECL) were obtained from Amersham Biosciences (Piscataway, NJ). EZ-Chemiluminescence kit was obtained from Biological Industries (Kibbutz Beit Haemek, Isreal). Antibodies against CYP5122A1 and CYP710C1 were raised in rabbits against peptides specific for the proteins by Abexome Bioscience (Bangalore, India). Horse Radish Peroxidase (HPR) -conjugated goat anti-rabbit IgG and goat anti rat IgG were obtained from Jackson Immuno Research Laboratories Inc. (West Grove, PA USA). Alexa Flour® 488 and Alexa Fluor® 594 conjugated goat anti-rabbit IgG were obtained from Invitrogen (Carlsbad, CA). Rabbit anti-Tubulin-a and anti-Tubulin-p antibodies were obtained from Neomarker (Fremont, CA) Anti-BIP antibody was obtained as a kind gift from Dr. J. D. Bangs, University of Wisconsin (Madison, USA). Mouse monoclonal IgG anti-GAPDH antibody was from Santa Cruz Biotechnology Inc., CA. Normal Goat Serum (NGS) was procured from GIBCO BRL (Gaithersburg, MD). MitoTracker® Red, MitoTracker® Green FM, ERTracker™ Blue White DPX, JC-1 (5S,6,6'-tetrachloro-1X3,3'-tetraethylbenzimidazolyl carbocyanine iodide), Rhod-2,AM, CM-H2DCFDA (5-(and-6) chloromethyl-2' ,7' -dichlorodihydrofluoroscein diacetate, acetyl ester), Syto 11 green nucleic acid stain, Pluronic F127, Hoechst, ATP determination kit, Nonyl Aridine Orange (NAO) were obtained from Molecular Probes (Eugene, OR). Propidium iodides, Giemsa stain, Peanut agglutinin, Monodansyl Cadaverine, MIT reagent (Thiozolyl blue tetrazolium bromide) were obtained from Sigma Aldrich (StLouis, MO).

Dulbecco' s Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute medium (RPMI-1640), Phenol red free DMEM, and Medium 199 (M199) were purchased from Sigma Aldrich (St. Louis, MO). Gentamycin was procured from GIBCO BRL (Gaithersburg, MD). Foetal Bovine Serum (FBS) was purchased from Biological Industries Ltd. (Kibbutz Beit Haemek, Isreal). Brain Heart Infusion Agar was obtained from Pronadisa (CONDA Laboratories, Madrid, Spain). LB agar was obtained from Invitrogen Technologies (Carlsbad, CA) while LB broth was obtained from Himedia (India). Sodium bicarbonate, HEPES buffer, Hemin, Biotin, Glucose, Adenine, Xanthine, Tween 80, DMSO and Triethanolamine were obtained from Sigma Aldrich (St Louis, MO). Potassium Antimony Tartrate (PAT), Kanamycin, Ampicillin, and Ergosterol were obtained from Sigma Aldrich (StLouis, MO). Miltefosine was obtained from Cayman Chemicals (Ann Arbor, MI). Hydrogen Peroxide (H202) was obtained from Merck Specialities Pvt. Ltd. (Mumbai, India). Hygromycin B was obtained from Sigma Aldrich (St. Louis, MO) and Invitrogen (Carlsbad, CA). Deoxy-ribonucleotide ( dNTP) mix, Magnesium chloride (MgCh), and pGEM-TEasy vector were obtained from Promega (Madison, WI). Taq DNA Polymerase and all restriction enzymes used were procured from New England BioLabs (Beverley, MA). 1 Kb DNA Ladder, 6X Gel Loading dye were obtained from MBI Fermentas (Ontario, Canada). Synthetic oligo-nucleotides were obtained from Sigma GENOSYS (Bangalore, India) and Microsynth (Germany). Platinum High Fidelity Taq Polymerase (HiFi Taq), Superscript Reverse Transcriptase, oligo dT12-1s, Dithiothretol (DTT), RNase OUTfM, RNase Hand Trizol® were procured from Invitrogen Technologies (Carlsbad, CA). DEPC, Agarose, Calcium chloride (CaCh), Blue White Select Screening Reagent, Proteinase K, Ethidium bromide, were obtained from Sigma Aldrich (St. Louis, MO). Low melting agarose (ultrapure grade) was procured from United States ~iochemical (Cleveland, OH). Plasmid MiniPrep kit, MidiPrep kit and EndoFree Maxiprep kit, PCR Purification kit and Gel Extraction kit were obtained from Qiagen (GmbH, Hilden). GenElute™ Mammalian Genomic DNA MiniPrep kit was obtained from Sigma Aldrich (St. Louis, MO)

MATERIALS