Acanthamoeba castellanii and human corneal epithelial (HCE) cells were obtained from the American Type Culture Collection (ATCC 30868 and ATCC PCS-700–010, respectively). Fusarium solani (NCCP 32678), Pseudomonas aeruginosa (NCCP 16091), and Staphylococcus aureus (NCCP 15920) were obtained from the Korea Centers for Disease Control and Prevention. Acanthamoeba trophozoites were cultured axenically in peptone-yeast-glucose (PYG) medium at 25 °C, and Acanthamoeba cysts were induced in encystment media (95 mM NaCl, 5 mM KCl, 8 mM MgSO4, 0.4 mM CaCl2, 1 mM NaHCO3 and 20 mM Tris-HCl, pH 9.0) at 25 °C for 2 days [26 (link)]. HCE cells were cultured at 37 °C with 5% CO2 in keratinocyte growth medium (KGM; Lonza, Portsmouth, NH, USA). F. solani was cultured in Sabouraud Dextrose (SD) media at 25 °C, and P. aeruginosa and S. aureus were cultured in Brain Heart Infusion (BHI) media at 37 °C.
Acanthamoeba castellanii
Manufactured by American Type Culture Collection
Sourced in United States
Acanthamoeba castellanii is a species of free-living amoeba commonly found in soil, fresh water, and air. It is a type of protist that is useful for various research applications, such as studies on microbial ecology, cellular biology, and host-pathogen interactions.
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5 protocols using acanthamoeba castellanii
1
Standardizing Acanthamoeba and Ocular Pathogen Cultivation
2
Vibrio cholerae OmpA Mutant Analysis
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The bacterial strains used in this study were wild-type Vibrio cholerae strain A1552 O1 El Tor Inaba [18 (link)] and its OmpA mutant by internal in-frame deletion of OmpA gene, kindly provided by Dr. S. N. Wai, University of Umeå. It has been proven that the OmpA mutant bacterium fails to produce OmpA protein, unlike the wild-type strain [7 (link)]. Acanthamoeba castellanii was obtained from the American Type Culture Collection (ATCC 30234), Manassas, VA, USA.
V. cholerae strains were stored frozen in Luria-Bertani (LB) medium with 15% glycerol at −80°C. Both bacterial strains were grown overnight at 37°C on LB plates and thereafter in LB broth, with shaking to an absorbance600 of 0.6. A. castellanii was grown without shaking at 30°C to a final concentration of 106/mL in ATCC medium number 712.
V. cholerae strains were stored frozen in Luria-Bertani (LB) medium with 15% glycerol at −80°C. Both bacterial strains were grown overnight at 37°C on LB plates and thereafter in LB broth, with shaking to an absorbance600 of 0.6. A. castellanii was grown without shaking at 30°C to a final concentration of 106/mL in ATCC medium number 712.
3
Culturing and Enumerating Acanthamoeba castellanii
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Acanthamoeba castellanii were purchased from the American Type Culture Collection (ATCC 50492). Amoebae were cultured in PYG (0.75% protease peptone, 0.75% yeast, and 1.5% glucose) at 30 °C in T-75 flasks as described previously [9 (link),11 (link),32 (link),33 (link),34 (link)]. In short, upon confluency, flasks were placed on ice for 10 min, with gentle tapping. Non-adherent amoebae were collected in 15 mL tubes and centrifuged at 2700× g for 5 min. Next, amoebae were enumerated using a hemocytometer and used for various experiments.
4
Culturing Acanthamoeba and Human Cells
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Amoeba and cell culture Acanthamoeba castellanii (strain ATCC30011) was obtained from the American Type Culture Collection (ATCC). Trophozoites were cultured axenically in peptone-yeast-glucose (PYG) medium. Cultures were incubated at 26 °C and trophozoites were harvested in the late log phase after subculture for 72 h. HEp-2 cells purchased from ATCC were cultured in EMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Thermo), 100 U/ml penicillin and 100 µg/ml streptomycin. Human corneal epithelial cells (HCECs) purchased from Bioleaf (Shanghai, China) were grown in DMEM (Gibco) with 10% FBS. Both cells were cultured in a 37 °C incubator with 5% CO2.
5
Culturing L. pneumophila and E. coli for Research
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All L. pneumophila strains were cultured on buffered charcoal yeast extract (BCYE) plates, or in N-(2-acetamido)-2-aminoethanesulfonic acid–buffered yeast extract (AYE) medium, supplemented with thymidine (100 μg/ml) (63 (link)) when required. E. coli DH5α used as host strains for cloning strategies was grown in LB and agar at 37 °C. For liquid culture, AYE broth was inoculated with TP bacteria grown in the previous cycle to a final absorbance of 0.2 at 600 nm and incubated at 37 °C with vigorous shaking. RP bacteria were harvested at an absorbance of 0.7 to 1.0 at 600 nm, and TP bacteria were harvested approximately 6 h after the cessation of growth, which is at an approximate absorbance of 3.0 to 3.5 at 600 nm, according to the one previously reported (11 (link), 61 (link)). Acanthamoeba castellanii (American Type Culture Collection 30234) was grown in proteose yeast extract glucose medium at 30 °C (64 (link)). To ascertain colony-forming units (CFU), serial dilutions of bacteria were incubated on BCYE for 4 days and resultant colonies were counted. The bacterial strains, plasmids, and primers used in this work are listed in supplemental Tables S18 and in S19 in the supplementary data, respectively.
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