Newly transformed schistosomula (NTS) were maintained for 2 h in M169 (Vitrocell) medium supplemented with 2% fetal bovine serum (FBS) (Vitrocell), 1 μM serotonin, 0.5 μM hypoxanthine,1 μM hydrocortisone, 0.2 μM triiodothyronine, penicillin/streptomycin, amphotericin, gentamicin (Vitrocell) at 37°C and 5% CO2 [28 (link)]. Only after 2 h incubation in culture medium the NJ series treatment was initiated as follows: 6.25 μM, 12.5 μM, 25 μM or 50 μM compound, for 1–5 days. Paired adult worms freshly perfused from infected hamsters (see above) were maintained in culture in RPMI medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Vitrocell), penicillin/streptomycin, amphotericin, gentamicin (Vitrocell) at 37°C and 5% CO2 for 2 h prior to the beginning of treatment with NJ series compound as follows: 12.5 μM, 25 μM or 50 μM compound, for 1–3 days of treatment. In all cases, NJ series compounds were prepared from a stock solution of 20 mM in dimethyl sulfoxide (DMSO), and the equivalent amount of DMSO was added to the control assays.
Gentamicin
Manufactured by Vitrocell
Sourced in Brazil
Gentamicin is a laboratory product used for cell culture applications. It is an antibiotic that inhibits bacterial growth, which helps maintain the sterility of cell culture environments.
Automatically generated - may contain errors
Lab products found in correlation
Amphotericin, by Vitrocell (5 mentions)
Penicillin streptomycin, by Vitrocell (4 mentions)
Rpmi medium, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Vitrocell (3 mentions)
Qiaamp viral rna extraction kit, by Qiagen (2 mentions)
Amphotericin b, by Vitrocell (2 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Trichostatin a, by Cayman Chemical (1 mentions)
L glutamine, by Merck Group (1 mentions)
8 protocols using gentamicin
1
Schistosoma Parasite Culturing and Treatment
2
Schistosome Life Cycle Stage-Specific Treatment
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Schistosomula, juvenile worms and adult worms were treated with different final concentrations of GSK343 in culture medium specific to each stage as indicated in the Results (from a stock solution of 20 mM GSK343 in DMSO), and with the equivalent amount of DMSO in the control assays. Newly transformed schistosomula (NTS) were maintained for 16 h in M169 (Vitrocell) medium supplemented with 2% fetal bovine serum (FBS) (Vitrocell), 1 μM serotonin, 0.5 μM hypoxanthine,1 μM hydrocortisone, 0.2 μM triiodothyronine, penicillin/streptomycin, amphotericin, gentamicin (Vitrocell) at 37°C and 5% CO2 [25 (link)]. Only after 16h incubation with the culture medium was the GSK343 treatment initiated. Juvenile worms and paired adult worms were maintained in RPMI medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Vitrocell), penicillin/streptomycin, amphotericin, gentamicin (Vitrocell) at 37°C and 5% CO2.
3
Isolation and Maintenance of Schistosomula
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Cercariae shed from infected snails were cooled on ice for 30 min and collected by centrifugation. Schistosomula were obtained by mechanical transformation of cercariae, and separation of their bodies as previously described72 (link). The newly transformed schistosomula (NTS) were maintained for 72 h, before coculture, in M169 medium (Vitrocell, cat number 00464) supplemented with penicillin/streptomycin, amphotericin, gentamicin (Vitrocell, cat number 00148), 1 µM serotonin, 0.5 µM hypoxanthine, 1 µM hydrocortisone and 0.2 µM triiodothyronine at 37 °C and 5% CO2.
4
Alphavirus Propagation and RNA Extraction
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The alphaviruses used for evaluation of the real-time RT-PCR were propagated in C6/36 Aedes albopictus cells cultured in Leibovitz's L-15 medium supplemented with 10 % heat-inactivated fetal bovine serum, 50 mg/ml of gentamicin and 2 mg/ml of amphotericin B (Vitrocell, Brazil). The infected cells were incubated for three days at 28 °C. Cell infection with alphavirus was confirmed by an indirect immunofluorescent test, as previously described [6] . The culture medium of infected cells was centrifuged, and the supernatant was aliquoted as viral stock and stored at -80 °C. Viral RNA was extracted using the QIAamp viral RNA extraction kit (Qiagen, Germany) and the viral RNA from alphavirus was converted to double-stranded cDNA using Superscript III (Invitrogen, USA), both according to the manufacturer's instructions.
5
Flavivirus Propagation and RNA Extraction
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The flaviviruses used for evaluation of the real-time RT-PCR were propagated in C6/36 Aedes albopictus cells cultured in Leibovitz's-15 medium supplemented with 10% heat-inactivated fetal bovine serum, 50mg/mL of gentamicin, and 2mg/mL of amphotericin B (Vitrocell, Brazil). The infected cells were incubated for 5 to 10 days at 28°C. Next, viral RNA was extracted from the flaviviruses using the QIAamp viral RNA extraction kit (Qiagen, Germany) and converted to double-stranded complementary DNA (cDNA) using Superscript III reverse transcriptase (Invitrogen, USA), according to the manufacturer's instructions.
6
Schistosoma mansoni Maintenance and Exposure to Trichostatin A
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S. mansoni is maintained in the laboratory using the intermediate snail host Biomphalaria glabrata and as definitive host the golden hamster (Mesocricetus auratus). Cercariae were released from infected snails and mechanically transformed to obtain schistosomula in vitro [22 (link)]. Newly transformed schistosomula were maintained for 12 h in M169 (Vitrocell) medium supplemented with 2% fetal bovine serum (FBS) (Vitrocell), 1 μM serotonin, 0.5 μM hypoxanthine, 1 μM hydrocortisone, 0.2 μM triiodothyronine, penicillin/streptomycin, amphotericin, gentamicin (Vitrocell) at 37°C and 5% CO2 [23 (link)], after which time the drug treatment was initiated, as described below. Adult worms were obtained from 7-week infected hamsters by left ventricular perfusion, and release of worms from the hepatic portal vein. Paired worms were maintained in RPMI medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Vitrocell), penicillin/streptomycin, amphotericin (Vitrocell) at 37°C and 5% CO2.
The parasites were treated with 1 μM Trichostatin A (Cayman Chemical), a concentration that has been shown by Dubois et al. [15 (link)] to be sub-lethal, and the negative controls with an equivalent amount of ethanol (vehicle of TSA), for 12, 24 and 48 h for microarray experiments, and for 12 h for ChIP-qPCR and western-blotting experiments.
The parasites were treated with 1 μM Trichostatin A (Cayman Chemical), a concentration that has been shown by Dubois et al. [15 (link)] to be sub-lethal, and the negative controls with an equivalent amount of ethanol (vehicle of TSA), for 12, 24 and 48 h for microarray experiments, and for 12 h for ChIP-qPCR and western-blotting experiments.
7
Embryonic Cell Line Culture Protocol
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The embryonic cell line from A. sculptum IBU/ASE-16 [24 ] was cultured in commercial Leibovitz L-15 culture medium containing gentamicin sulfate and amphotericin (Vitrocell Embriolife, Campinas, SP, Brazil) and supplemented with 0.01% L -glutamine (Sigma‒Aldrich, St. Louis, MO, USA), as previously described [17 (link)].
8
Schistosoma LSD1 Inhibitor Assay
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Schistosomula or adult worms were treated with different concentrations of LSD1 inhibitors, as indicated in the figure legends. For each treatment condition, 10 worm pairs were maintained in 60-mm diameter culture dishes in 2 mL of culture medium (medium M169 (Gibco) supplemented with 10% fetal bovine serum (Vitrocell), penicillin/streptomycin, amphotericin and gentamicin (Vitrocell). Schistosomula were maintained in 96-well or 24-well culture plates, depending on the experiment, with 200 μL or 1 mL of culture medium M169 (Gibco), respectively, supplemented with 2% fetal bovine serum (Vitrocell), 1 μM serotonin, 0.5 μM hypoxanthine, 1 μM hydrocortisone, 0.2 μM triiodothyronine, penicillin/streptomycin, amphotericin and gentamycin (Vitrocell). Parasites were maintained at 37°C in 5% CO2 with a humid atmosphere. The medium containing the LSD1 inhibitors or DMSO (vehicle) was refreshed every 24 h during the treatment period (1–4 days).
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