Dithiothreitol (DTT; Sigma-Aldrich, St. Louis, MO, USA), Basal Medium Eagle (BME) vitamins (Sigma-Aldrich, St. Louis, MO, USA), and pooled human urine (Innovative Research Inc., Peary Court Novi, MI, USA) were used.
Basal medium eagle vitamins
Manufactured by Merck Group
Sourced in United States
Basal Medium Eagle vitamins are a standardized mixture of vitamins commonly used as a supplement in cell culture media. The vitamins provided include essential nutrients required for the optimal growth and maintenance of various cell types in in vitro environments.
Automatically generated - may contain errors
Lab products found in correlation
M199 medium, by Merck Group (5 mentions)
Amikacin, by Bristol-Myers Squibb (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Pooled human urine, by Innovative Research (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Gentamicin sulfate, by Merck Group (1 mentions)
Schneider s insect medium, by Merck Group (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Amikacin amikin, by Bristol-Myers Squibb (1 mentions)
Neomycin, by Merck Group (1 mentions)
Amikin, by Bristol-Myers Squibb (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Biosera (1 mentions)
Biopterin, by Merck Group (1 mentions)
Hygromycin b, by Carl Roth (1 mentions)
7 protocols using basal medium eagle vitamins
1
Urine-Based Cell Culture Supplementation
2
Isolation and Cultivation of Leishmania orientalis Parasites
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L. orientalis parasites (MHOM/TH/2014/LSCM4) were originally isolated from a skin lesion on the face of Thai woman patient (Jariyapan et al. 2018) . The isolated parasites were initially grown as promastigotes in Schneider's insect medium (Sigma-Aldrich, St Louis, MO, USA), pH 6.8 supplemented with 20% (v/v) FCS (Life Technologies-Gibco, Grand Island, NY, USA) at 26 °C. Some were then cryopreserved as parasite culture stock at the Department of Parasitology, Faculty of Medicine, Chiang Mai University. As a routine promastigote culture, parasites were grown at 26 °C in M199 medium, pH 6.8 with Hank's balance salt solution (HBSS) (Hyclone, Logan, UT, USA) supplemented with 10% (v/v) FCS, 2% (v/v) healthy human urine, 1% (v/v) Basal Medium Eagle (BME) vitamins (Sigma-Aldrich, St Louis, MO, USA) and 25 μg/ml gentamicin sulfate (Sigma-Aldrich, St Louis, MO, USA). Promastigotes were subpassaged to fresh medium every 4 days to maintain growth and viability of the parasites.
3
Promastigote Cultivation and 18S rRNA Sequencing
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Promastigotes were cultured in the M199 medium (Sigma−Aldrich, St. Louis, MO, United States) containing 10% heat-inactivated fetal bovine calf serum (FBS; Thermo Fisher Scientific, Waltham, MA, United States), supplemented with 1% Basal Medium Eagle vitamins (Sigma−Aldrich), 2% sterile urine and 250 μg/ml of amikacin (Bristol-Myers Squibb, New York, NY, United States).
Total genomic DNA was isolated from 10 ml of trypanosomatid cultures with the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. 18S rRNA gene was amplified using primers S762 and S763 [48 (link)], following the previously described protocol [13 (link)]. These PCR fragments were sequenced directly at Macrogen Europe (Amsterdam, Netherlands) as described previously [49 (link)]. The identity of species under study was confirmed by BLAST analysis [50 (link)].
Total genomic DNA was isolated from 10 ml of trypanosomatid cultures with the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. 18S rRNA gene was amplified using primers S762 and S763 [48 (link)], following the previously described protocol [13 (link)]. These PCR fragments were sequenced directly at Macrogen Europe (Amsterdam, Netherlands) as described previously [49 (link)]. The identity of species under study was confirmed by BLAST analysis [50 (link)].
4
Cultivation of Leishmania and Herpetomonas
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Leishmania donovani (MHOM/ET/2010/GR374), L. major LV561 (LRC-L137; MHOM/IL/1967/Jericho-II) and Herpetomonas muscarum [11 (link)] were cultured in M199 medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% heat-inactivated foetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1% Basal Medium Eagle vitamins (Sigma-Aldrich), 2% sterile urine, 250 μg/ml amikacin (Amikin; Bristol-Myers Squibb, Princeton Pike, NJ, USA) at 23 °C (L. donovani, L. major) or 28 °C (H. muscarum).
5
Cultivation of Leishmania infantum mCherry Promastigotes
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Leishmania infantum mCherry (MHOM/TR/2000/OG-VL) promastigotes (transfected with red fluorescence protein) were cultivated in M199 medium (Sigma) containing 20% heat-inactivated fetal bovine serum (FBS) (Gibson), supplemented with 2% sterile urine, 1% Basal Medium Eagle vitamins (Sigma), 250 µg mL−1 amikacin (Amikin, Bristol-Myers Squibb) and 150 µg mL−1 selective antibiotic neomycin (Sigma). Each week parasites were passaged to the fresh medium, during all experiments, parasites with in-vitro passage number less than 10 were used. For experimental infection in IRD Montpellier, two vials containing frozen L. infantum mCherry (MHOM/TR/2000/OG-VL) were shipped on dry ice from CUNI, 15 d before the first experimental infection, allowing the IRD hosts to establish stable cultures of parasites.
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Leishmania infantum mCherry (MHOM/TR/2000/OG-VL) promastigotes (transfected with red fluorescence protein) were cultivated in M199 medium (Sigma) containing 20% heat-inactivated fetal bovine serum (FBS) (Gibson), supplemented with 2% sterile urine, 1% Basal Medium Eagle vitamins (Sigma), 250 µg mL−1 amikacin (Amikin, Bristol-Myers Squibb) and 150 µg mL−1 selective antibiotic neomycin (Sigma). Each week parasites were passaged to the fresh medium, during all experiments, parasites with in-vitro passage number less than 10 were used. For experimental infection in IRD Montpellier, two vials containing frozen L. infantum mCherry (MHOM/TR/2000/OG-VL) were shipped on dry ice from CUNI, 15 d before the first experimental infection, allowing the IRD hosts to establish stable cultures of parasites.
6
Culturing L. mexicana Promastigotes and Differentiating Metacyclic and Amastigote Forms
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L. mexicana (MNYC/BZ/1962/M379) promastigotes were cultured in M199 medium (Sigma-Aldrich, St. Louis, USA), supplemented with 2 µg/ml biopterin (Sigma-Aldrich), 2 µg/ml Hemin (Jena Bioscience GmbH, Jena, Germany), 25 mM HEPES and 50 units/ml of penicillin/streptomycin (Life Technologies/Thermo Fisher Scientific, Carlsbad, USA), and 10% heat-inactivated fetal bovine serum (FBS, BioSera Europe, Nuaillé, France). Metacyclic promastigotes and amastigotes were differentiated as previously described [44 (link)]. Prior to in vivo analysis, Leishmania cell lines were passaged through mice. Freshly isolated promastigotes were cultured in M199 medium (Sigma-Aldrich) containing 10% heat-inactivated FBS (Life Technologies) supplemented with 1% Basal Medium Eagle vitamins (Sigma-Aldrich), 2% sterile human urine, and 250 μg/ml amikacin (Bristol-Myers Squibb, New York, USA). For mice infections, the pH of the cultivation medium was adjusted to 5.5.
7
Culturing L. mexicana Promastigotes and Evaluating Macrophage Infection
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The L. mexicana wild type promastigotes were cultured in the M199 medium (Sigma-Aldrich) containing 10% FBS supplemented with 1% Basal Medium Eagle vitamins (Sigma-Aldrich), 2% sterile urine, and 250 µg/mL Amikacin (Bristol-Myers Squibb, New York, NY, USA), and 200 µg/mL of Hygromycin B (Carl Roth GmbH) for BnSP-7-ecDHFR-HA mutant cell lines. Infection of the J774 macrophages was performed as described previously [49 (link),52 (link)]. In addition, infection was evaluated by counting Giemsa-stained slides as described previously [51 (link)]. All experiments were performed with two independent biological replicates and samples were analyzed in triplicate.
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