Leishmania parasites were previously isolated from a patient with mucosal leishmaniasis at Corte de Pedra, Bahia, Brazil, and characterized as L. (V.) braziliensis by the Leishmania Collection at Fundação Oswaldo Cruz—CLIOC/FIOCRUZ. To preserve the infectivity, the parasites were inoculated via intraperitoneal route and maintained through regular passages in hamster (Mesocricetus auratus). Amastigotes were then purified from the spleen of hamster and expanded in axenic culture with Schneider’s insect medium (Sigma-Aldrich, USA) containing 100 UI/ml penicilin and 100 μg/ml streptomycin and supplemented with 10% heat-inactivated fetal calf serum (FCS) (Cultilab, Brazil) at 26°C. The amastigote-derived promastigotes were cryopreserved in aliquots and thawed for use in specific experiments. Promastigotes were cultured in Schneider’s insect medium (Sigma-Aldrich, USA) containing 100 UI/ml penicilin and 100 μg/ml streptomycin and supplemented with 10% heat-inactivated fetal calf serum (FCS) (Cultilab, Brazil) at 26°C. The parasites used in the experiments were at the stationary phase of growth and with no more than four passages in culture.
Schneider s insect medium
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany, Brazil, Sao Tome and Principe, Macao, France
Schneider's insect medium is a laboratory culture medium designed for the growth and maintenance of insect cells. It provides the necessary nutrients and growth factors for the cultivation of various insect cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (46 mentions)
Streptomycin, by Merck Group (24 mentions)
Penicillin, by Thermo Fisher Scientific (22 mentions)
Penicillin, by Merck Group (18 mentions)
Streptomycin, by Thermo Fisher Scientific (15 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions)
Fetal bovine serum (fbs), by Merck Group (9 mentions)
Fetal bovine serum (fbs), by Cultilab (7 mentions)
L glutamine, by Thermo Fisher Scientific (7 mentions)
Gentamicin, by Merck Group (6 mentions)
Phenylthiourea ptu, by Merck Group (6 mentions)
Hemin, by Merck Group (6 mentions)
Penicillin streptomycin, by Merck Group (6 mentions)
Gentamicin, by Thermo Fisher Scientific (5 mentions)
6 biopterin, by Merck Group (4 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (4 mentions)
Biopterin, by Merck Group (4 mentions)
Gentamicin, by Merck & Co (4 mentions)
Effectene transfection reagent, by Qiagen (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
223 protocols using schneider s insect medium
1
Leishmania braziliensis Isolation and Propagation
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Leishmania parasites were previously isolated from a patient with mucosal leishmaniasis at Corte de Pedra, Bahia, Brazil, and characterized as L. (V.) braziliensis by the Leishmania Collection at Fundação Oswaldo Cruz—CLIOC/FIOCRUZ. To preserve the infectivity, the parasites were inoculated via intraperitoneal route and maintained through regular passages in hamster (Mesocricetus auratus). Amastigotes were then purified from the spleen of hamster and expanded in axenic culture with Schneider’s insect medium (Sigma-Aldrich, USA) containing 100 UI/ml penicilin and 100 μg/ml streptomycin and supplemented with 10% heat-inactivated fetal calf serum (FCS) (Cultilab, Brazil) at 26°C. The amastigote-derived promastigotes were cryopreserved in aliquots and thawed for use in specific experiments. Promastigotes were cultured in Schneider’s insect medium (Sigma-Aldrich, USA) containing 100 UI/ml penicilin and 100 μg/ml streptomycin and supplemented with 10% heat-inactivated fetal calf serum (FCS) (Cultilab, Brazil) at 26°C. The parasites used in the experiments were at the stationary phase of growth and with no more than four passages in culture.
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Leishmania parasites were previously isolated from a patient with mucosal leishmaniasis at Corte de Pedra, Bahia, Brazil, and characterized as L. (V.) braziliensis by the Leishmania Collection at Fundação Oswaldo Cruz—CLIOC/FIOCRUZ. To preserve the infectivity, the parasites were inoculated via intraperitoneal route and maintained through regular passages in hamster (Mesocricetus auratus). Amastigotes were then purified from the spleen of hamster and expanded in axenic culture with Schneider’s insect medium (Sigma-Aldrich, USA) containing 100 UI/ml penicilin and 100 μg/ml streptomycin and supplemented with 10% heat-inactivated fetal calf serum (FCS) (Cultilab, Brazil) at 26°C. The amastigote-derived promastigotes were cryopreserved in aliquots and thawed for use in specific experiments. Promastigotes were cultured in Schneider’s insect medium (Sigma-Aldrich, USA) containing 100 UI/ml penicilin and 100 μg/ml streptomycin and supplemented with 10% heat-inactivated fetal calf serum (FCS) (Cultilab, Brazil) at 26°C. The parasites used in the experiments were at the stationary phase of growth and with no more than four passages in culture.
2
Puromycin Labeling of Nascent Proteins
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For puromycin labeling experiments, tissues were dissected in Schneider's insect medium (Sigma-Aldrich, USA) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich) at 25°C. They were then incubated with Schneider's insect medium containing puromycin (5 μg/ml, Sigma-Aldrich) and cycloheximine (100 µg/ml, Sigma-Aldrich) for 2 h at RT. Then the samples were fixed with 4% PFA in PBST 0.2% at RT and blocked overnight with blocking buffer at 4°C. Primary anti-puromycin antibody (diluted in PBST) was incubated with the samples for 8 h at RT. The samples were rinsed three times and washed three times (20 min/wash) with PBST. Secondary antibodies (diluted in PBST) were incubated overnight at 4°C. The samples were then rinsed three times and washed two times (20 min/wash) with PBST. Hoechst 33258 (2.5 μg/ml) was added in PBST before the last washing step and the samples were mounted with Aqua/Poly Mount solution (Polysciences). See Table S2 for antibody details.
3
H9N2 Virus Propagation, Inactivation, and Purification
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A/Chicken/Pakistan/UDL 01/2008 (UDL01/08) H9N2 virus was propagated in 10-day-old SPF embryonated hens’ eggs and titrated by plaque assay or TCID50 (50% tissue culture infective dose) on Madin Darby canine kidney (MDCK) cells. The virus was inactivated chemically using 0.1% beta-propiolactone (BPL, Alfa Aesar) and purified by ultracentrifugation through a continuous 30−60% w/v sucrose gradient.
MDCK cells were maintained in Dulbecco modified eagle medium (DMEM, Merck Life Science) supplemented with 10% fetal bovine serum (FBS) and 0.1% penicillin and streptomycin at 37 °C, 5% CO2. Drosophila melanogaster Schneider 2 (S2) cells were obtained from Thermofisher Scientific and maintained in Schneider’s insect medium (Merck Life Science) supplemented with 10% FBS at 25 °C.
MDCK cells were maintained in Dulbecco modified eagle medium (DMEM, Merck Life Science) supplemented with 10% fetal bovine serum (FBS) and 0.1% penicillin and streptomycin at 37 °C, 5% CO2. Drosophila melanogaster Schneider 2 (S2) cells were obtained from Thermofisher Scientific and maintained in Schneider’s insect medium (Merck Life Science) supplemented with 10% FBS at 25 °C.
4
Versatile Cell Culture and Transfection Protocol
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The 293A, Vero, HeLa, Huh7, SH-SY5Y, 293T-derived knockout cell lines [82 (link)], and HCT116-derived knockout cell lines [68 (link)] were cultured in Dulbecco’s modified Eagle medium (DMEM)-high glucose (Nacalai Tesque, Kyoto, Japan) containing 100 units/mL penicillin, 100 μg/mL streptomycin, and 10% (v/v) fetal calf serum (FCS), in a humidified atmosphere containing 5% CO2 at 37° C. C6/36 cells were cultured in Schneider’s insect medium (Merck KGaA, Darmstadt, Germany) containing 100 units/mL penicillin, 100 μg/mL streptomycin, and 10% (v/v) FCS at 25°C. The JEV AT31 strain (kindly provided by Eiji Konishi, Osaka University, Japan) was propagated in 293A cells. PEI MAX (Polysciences, Warrington, PA, USA) or Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) were used for transfection according to the manufacturer’s protocols.
5
Leishmania aethiopica Parasite Cultivation
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Two L. guyanensis clones, designated here as Lg M4147 LRV1+ and Lg M4147 LRV1−, and which were previously shown to be highly- and non-infected by LRV respectively (designated as LRVhigh and LRVneg in [27] (link)), were used as reference parasites.
Eight strains of L. aethiopica parasites were freshly isolated from infected patients who contracted leishmaniasis in Ethiopia (Table 1 , fresh isolates). In addition, three L. aethiopica lines from Leishmania species reference centers were also used (Table 1 , cryobank lines), and kindly provided by Charles Jaffe and Lee Schnur (Jerusalem, Israel).
All parasite strains were cultivated as promastigotes at 26°C in Schneider's insect medium (Sigma) supplemented with fetal bovine serum, Hepes, penicillin/streptomycin, biopterin and hemin as described before [28] (link).
Eight strains of L. aethiopica parasites were freshly isolated from infected patients who contracted leishmaniasis in Ethiopia (
All parasite strains were cultivated as promastigotes at 26°C in Schneider's insect medium (Sigma) supplemented with fetal bovine serum, Hepes, penicillin/streptomycin, biopterin and hemin as described before [28] (link).
6
Culturing Cell Lines and Primary Mouse Fibroblasts
7
Calcium Dynamics During Insect Ecdysis
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Calcium (Ca2+) recordings were carried out essentially as described in Mena et al. [25 (link)]. Briefly, animals containing a bubble in the mid-region of the puparium (~4 hours before pupal ecdysis) were selected. The central nervous system (CNS) was dissected in cold PBS, immobilized in 1.5% low melting temperature agarose solution (Sigma type VII-A; Sigma-Aldrich Chemical Co., MO) and covered with Schneider’s Insect Medium (Sigma-Aldrich Chemical Co., MO). Recordings were performed using an Olympus DSU Spinning Disc microscope (Olympus Corporation, Shinjuku-ku, Tokyo, Japan) under a 20 X W NA 0.50 immersion lens. GFP signal was acquired using an ORCA IR2 Hamamatsu camera (Hamamatsu Photonics, Higashi-ku, Hamamatsu City, Japan) using the CellR Olympus Imaging Software (Olympus Soft Imaging Solutions, Munich, Germany). Fictive ecdysis was triggered by adding 600 nM of ETH1 (Bachem Co., USA). We recorded multiplane fluorescence using a sampling rate of 1 picture every 2–3 second for at least 60 min. Depending on the preparation, the number of images per z-stack was 3–5 focal planes (covering 100–200 μm in depth), which allowed the entire motoneuronal and CCAP network to be imaged.
8
Culturing Leishmania amazonensis Promastigotes
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Parasites of L. amazonensis (MHOM/Br88/Ba-125) were obtained from the Laboratory of Host - Parasite Interaction and Epidemiology - Gonçalo Moniz Institute (Bahia-Brazil). Axenic L. amazonensis promastigotes were grown in Schneider’s Insect Medium (Sigma, St. Louis, MO, USA) supplemented with 50 μg/mL gentamicin (Gibco, Grand Island, NY, USA) and 10% fetal bovine serum (Gibco, Grand Island, NY, USA). Cultures were maintained in an incubator at 24 °C until in vitro experiments were performed.
9
Culturing and Characterizing S2-ATCC Cells
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S2-ATCC cells (RRID: CVCL_Z232 ) was obtained from American Type Culture Collection (ATCC). Its identity was confirmed by visual inspection of the cell morphology and its growth kinetics in Schneider's insect medium (Sigma)/10% fetal bovine serum (FBS). A mycoplasma test is usually not done for S2 cells (Berndt et al., 2017 (link)).
The cell numbers were counted by using Luna automated cell counter (Logos Biosystems, Anyang-si, South Korea), according to the manufacturer’s instruction.
The cell numbers were counted by using Luna automated cell counter (Logos Biosystems, Anyang-si, South Korea), according to the manufacturer’s instruction.
10
Culturing of Wing Discs
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Wing discs were dissected from third instar larvae in Schneider’s insect medium (Sigma-Aldrich) and a small fragment was removed with tungsten needles. Discs were cultured in Schneider’s insect medium supplemented with 2% heat activated fetal calf serum, 2.5% fly extract and 5 μg/ml insulin, for different periods of time (from 1 to 10 hours) at 25°C. Ex vivo images were taken using a Leica SPE confocal microscope and processed with Fiji software.
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