Dulbecco s modified eagle s medium 10 dmem

collagen type I hydrogels were synthesized from rat tail collagen as described previously.²⁸ (link) Briefly, reconstitution buffer (RB) was made by dissolving NaHCO₃ and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) in DI-H₂O to a final concentration of 24 mg NaHCO₃/ml H₂O and 96 mg HEPES/ml H₂O and sterile filtered through 0.2 μm syringe filters (MilliporeSigma, Burlington, MA). Dulbecco's Modified Eagle's Medium (10× DMEM, Sigma-Aldrich, St. Louis, MO, USA) was also sterile filtered. To generate sufficient hydrogel volume for five devices, 400 ml 2.5 mg collagen type I/ml total solution was prepared by mixing 22.8 ml of 10× DMEM, 22.8 ml of RB, 7.4 ml of 1 M NaOH, 120.7 ml of PBS, 206 ml of 4.39 mg/ml collagen type I from rat tail (Corning, NY, USA), and 22.8 ml of AlexaFluor 647-conjugated collagen type I (prepared as previously described^29,30 (link)). Collagen pre-polymer slurry was injected into microfluidic devices through gel ports [Fig. 1(c)], and devices were placed in 37 °C incubator for 30 min prior to hydration with PBS or cell culture medium. To limit evaporation, each device was placed in a 60 mm Petri dish (Falcon^™ Bacteriological, ThermoFisher, Waltham, MA, USA), containing a kimwipe (Kimberly-Clark, Irving, TX) soaked in PBS + 1% (vol/vol) penicillin–streptomycin (ThermoFisher).

Epimastigote forms of T. cruzi Dm28c strain were cultivated in liver infusion tryptose (LIT) medium pH 7.4 supplemented with 10% inactivated fetal bovine serum (Gibco) and 1% streptomycin/penicillin (Invitrogen), at 28°C. For the culturing of knockout parasites, 300 μg ml^–1 of hygromycin B (Invitrogen) and neomycin (G418 sulfate – Gibco) antibiotics were added to the culture medium. Rhesus monkey kidney monolayers cells (LLC-MK2) (Hull et al., 1962 (link)) were maintained in 10% Dulbecco’s modified Eagle’s medium (10% DMEM; Sigma Aldrich), containing 10% fetal bovine serum (Gibco), 200 U ml^–1 of penicillin, and 200 μg L^–1 of streptomycin sulfate. Metacyclic trypomastigotes obtained from axenic cultures of T. cruzi at stationary phase were used to initiate parasite intracellular life cycle in LLC-MK2 cells. Infection was performed in DMEM supplemented with 2% fetal bovine serum (Gibco), 200 U ml^–1 penicillin, and 200 μg ml^–1 streptomycin sulfate (2% DMEM). LLC-MK2 cultures were washed daily with PBS^+/+ buffer (NaCl 0.134 M, KCl 2.7 mM, Na₂HPO₄ 10 mM, KH₂PO₄ 1.8 mM, Ca²⁺ 0.9 mM, and Mg²⁺ 0.49 mM) to remove remaining epimastigotes. Released tissue-culture trypomastigotes (TCTs) were purified as described previously (Andrews et al., 1987 (link)) and used to maintain parasite intracellular life cycle and to perform all experiments involving these cells.