L. (V.) braziliensis (MHOM/BR/1975/M2903) promastigotes were maintained at 25°C, in Schneider's insect medium (Gibco, Thermo Fisher Scientific Inc, MA, USA) supplemented with 10% heat-inactivated fetal calf serum (Gibco, USA) and 200 IU of penicillin per mL, and 200 μg of streptomycin (Sigma Chemical Company, St. Louis, Mo, USA) per mL, and grown until stationary phase before using in Leishmania growth curve analysis. The stationary phase promastigotes were distributed in triplicate into 24-well plates (5 × 105 parasites/well) in a final volume of 1.0 mL of Schneider's insect medium (Gibco, USA) supplemented with 2% FCS and antibiotics, with or without 50 ng/mL recombinant human IGF-I (rIGF-I, R&D Systems, USA) and maintained during the experimental period.
Schneider s insect medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Schneider's insect medium is a cell culture medium designed for the growth and maintenance of insect cells. It provides the necessary nutrients and growth factors to support the in vitro cultivation of various insect cell lines.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Penicillin, by Thermo Fisher Scientific (5 mentions)
Schneider s insect medium, by Merck Group (3 mentions)
Effectene, by Qiagen (2 mentions)
Concanavalin a coated, by Merck Group (2 mentions)
Digital caliper, by Thermo Fisher Scientific (2 mentions)
Sh sy5y cell, by American Type Culture Collection (2 mentions)
Aedes albopictus c6 36 cells, by American Type Culture Collection (2 mentions)
Leibovitz s l 15 medium, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
A21206, by Thermo Fisher Scientific (2 mentions)
A31570, by Thermo Fisher Scientific (2 mentions)
Vectorshield, by Vector Laboratories (2 mentions)
A11122, by Thermo Fisher Scientific (2 mentions)
Ixonem em ccd camera, by Oxford Instruments (1 mentions)
L 15 medium, by Thermo Fisher Scientific (1 mentions)
49 protocols using schneider s insect medium
1
Culturing L. braziliensis Promastigotes
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L. (V.) braziliensis (MHOM/BR/1975/M2903) promastigotes were maintained at 25°C, in Schneider's insect medium (Gibco, Thermo Fisher Scientific Inc, MA, USA) supplemented with 10% heat-inactivated fetal calf serum (Gibco, USA) and 200 IU of penicillin per mL, and 200 μg of streptomycin (Sigma Chemical Company, St. Louis, Mo, USA) per mL, and grown until stationary phase before using in Leishmania growth curve analysis. The stationary phase promastigotes were distributed in triplicate into 24-well plates (5 × 105 parasites/well) in a final volume of 1.0 mL of Schneider's insect medium (Gibco, USA) supplemented with 2% FCS and antibiotics, with or without 50 ng/mL recombinant human IGF-I (rIGF-I, R&D Systems, USA) and maintained during the experimental period.
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L. (V.) braziliensis (MHOM/BR/1975/M2903) promastigotes were maintained at 25°C, in Schneider's insect medium (Gibco, Thermo Fisher Scientific Inc, MA, USA) supplemented with 10% heat-inactivated fetal calf serum (Gibco, USA) and 200 IU of penicillin per mL, and 200 μg of streptomycin (Sigma Chemical Company, St. Louis, Mo, USA) per mL, and grown until stationary phase before using in Leishmania growth curve analysis. The stationary phase promastigotes were distributed in triplicate into 24-well plates (5 × 105 parasites/well) in a final volume of 1.0 mL of Schneider's insect medium (Gibco, USA) supplemented with 2% FCS and antibiotics, with or without 50 ng/mL recombinant human IGF-I (rIGF-I, R&D Systems, USA) and maintained during the experimental period.
2
Drosophila S2 Cell Transfection and RNAi
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Drosophila S2 cells (provided by Shin-ichi Yanagawa, Kyoto University, Kyoto, Japan) (Schneider, 1972 (link); Yanagawa et al., 1998 (link)) were cultured in Schneider's Insect Medium (Gibco) supplemented with 10% FCS and antibiotics at 25°C. Transfection was performed using Effectene (Qiagen) according to the manufacturer's instructions, and cells were harvested 36-48 h after transfection.
For RNAi, dsRNA against lacZ (control) or IKKε (Oshima et al., 2006 (link)) was added to the medium to a final concentration of 37 nM, and 36-48 h later the cells were transferred and transfected with PKC53E-GFP as described above. Cells were harvested 36-48 h after plasmid transfection (3-4 days after dsRNA addition).
For immunofluorescence, cells were fixed in 4% PFA in PBS for 20 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 min, and blocked with 5% skimmed milk in TBS (Tris-buffered saline). Rabbit anti-GFP primary antibody and secondary antibodies were diluted (as indicated above) in blocking solution, and antibody incubation was performed for 1 h at room temperature. After each antibody incubation, the coverslips were washed three times with 0.1% Triton X-100 in PBS. The cells were mounted in Vectashield mounting medium.
For RNAi, dsRNA against lacZ (control) or IKKε (Oshima et al., 2006 (link)) was added to the medium to a final concentration of 37 nM, and 36-48 h later the cells were transferred and transfected with PKC53E-GFP as described above. Cells were harvested 36-48 h after plasmid transfection (3-4 days after dsRNA addition).
For immunofluorescence, cells were fixed in 4% PFA in PBS for 20 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 min, and blocked with 5% skimmed milk in TBS (Tris-buffered saline). Rabbit anti-GFP primary antibody and secondary antibodies were diluted (as indicated above) in blocking solution, and antibody incubation was performed for 1 h at room temperature. After each antibody incubation, the coverslips were washed three times with 0.1% Triton X-100 in PBS. The cells were mounted in Vectashield mounting medium.
3
Imaging S2 and HeLa Cells with Confocal Microscopy
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S2 cells were imaged in concanavalin A–coated (0.25 mg/ml; EMD Millipore) glass-bottom dishes (Ibidi) at 25°C in Schneider’s insect medium (Gibco) + 10% FBS (Invitrogen) using a 100× 1.4 NA Plan-Apochromatic differential interference contrast (DIC) objective lens (Carl Zeiss) mounted on an inverted microscope (TE2000U; Nikon) equipped with a CSU-X1 spinning-disk confocal head (Yokogawa Electric Corporation) and with two laser lines (488 nm and 561 nm). Images were detected with an iXonEM+ EM-CCD camera (Andor Technology), using the NIS-Elements software (Nikon). HeLa cells were imaged in the same system at 37°C in L15 medium + 10% FBS (Invitrogen). Images were analyzed in ImageJ, processed (contrast adjustments) in Photoshop CS4 (Adobe), and represent maximum intensity projections of multiple z stacks (step size: 0.5–1 µm).
4
Metaphase Arrest and EdU Labeling
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To enrich cells in metaphase testes were dissected and transferred to Schneider's insect medium (Sigma) containing MG132 (20 μM final concentration, Sigma-Aldrich) [52 (link)] or colcemid (100μM final concentration, Calbiochem) [31 (link)]. After 4.5h incubation with MG132 or colcemid at 25°C, GSCs/SGs arrested in metaphase with or without intact bipolar mitotic spindles, respectively. For EdU incorporation the dissected testes were incubated for 45min (10 μM, Thermofisher) in Schneider's insect medium (Gibco) at 25°C.
5
Culturing Prothoracic Glands and Brains
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In vitro cultures were set up in 24 well plates in Schneider’s insect medium (Gibco) supplemented with 10% (vol/vol) heat inactivated FBS (Sigma), 0.01% (vol/vol) Insulin solution (Sigma, I0516-5ML), and 1% (vol/vol) Pen/Strep (Sigma). 1 ug/ml of 20-hydroxy-ecdysone (Sigma) was added for control conditions. Dissections were performed at 48 hr and the prothoracic glands were removed prior to culture. Brains were cultured for 24 hr and the media was changed every 12 hr.
6
Culturing and Transfecting Drosophila S2 Cells
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Drosophila S2 cells were cultured in Schneider's Insect Medium (Gibco) supplemented with 10% FCS and antibiotics at 25°C (Schneider, 1972 (link)). pUAST vectors with actin5Ce-Gal4 drivers were cotransfected using Effectene (Qiagen) according to the manufacturer's instructions, and harvested 36-48 h after transfection. For immunofluorescence or time-lapse imaging, cells were replated on coverslips or glass-bottom dishes coated with Concanavalin A (Wako) and were allowed to spread for 1-2 h (Rogers et al., 2002 (link)). For drug treatments, cells were treated with 1 µM Latrunculin A (Wako) or 10 µM Colchicine (Wako) for 1 h before imaging. For immunofluorescence, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS (PBS-T) for 15 min, and blocked with 5% skimmed milk in Tris-buffered saline (TBS). Primary and secondary antibodies were diluted in the blocking solution. After each antibody incubation, the coverslips were washed three times with PBS-T. The cells were mounted in Vectashield mounting medium (Vector Labs).
7
Ovary culture with NPF and sNPF
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Adult females were cultured on standard medium and dissected in Schneider’s insect medium (Gibco). Approximately 6 ovaries were transferred to a microcentrifuge tube containing 20 μL Schneider’s medium supplemented with 15% fetal calf serum and 0.6% penicillin-streptomycin with the addition of NPF peptide, sNPF peptide, or PBS. Cultures were incubated at RT for 16 hours, and then samples were immunostained to count GSC number.
8
Testes Dissection and RNA FISH
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Testes from 2–3 day-old flies were dissected and transferred to Schneider’s insect medium (Gibco) with or without 50 ng/μl α-amanitin. After 2 hours of incubation at room temperature, testes were fixed in 4% formaldehyde in 1x PBS for 30 minutes and processed for RNA fluorescent in situ hybridization.
9
Drosophila Embryo Gastrulation Assay
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Two hours after egg deposition, Oregon R Drosophila embryos were collected and dechorionated in a 50% bleach solution before being incubated with either U73122 (20 μM) or U73343 (20 μM) for 30 min. Embryos were left to age for an additional hour in Schneider’s Insect Medium (Gibco) before fixation using a standard paraformaldehyde, heptane, and methanol procedure75 . Fixed embryos were then mounted using Slowfade Diamond with DAPI (Thermo Fisher Scientific). Embryos were visualised using an Olympus FV3000 and gastrulation assessed by key markers of the onset of gastrulation, marking primarily the formation of the ventral furrow and posterior midgut invagination.
10
Imaging Cellular Structures in S2 Cells
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S2 cells transiently expressing CD8-GFP were grown on concanavalin A–coated (0.5 mg/ml; EMD Millipore) glass coverslips at 25°C in Schneider’s insect medium (Gibco) + 10% FBS (Invitrogen) and fixed with 4% paraformaldehyde for 10 min and subsequently extracted with 0.1% Triton X-100 for 10 min. After short washes in PBS and blocking with 10% FBS, cells were incubated with mouse anti-lamin Dm0 (clone ADL67, 1:50; Developmental Studies Hybridoma Bank) and rat anti–α-tubulin (YOL1/34, 1:100; Serotec), followed by short washes in PBS and incubation with Alexa Fluor 568 and 647 (1:1,000; Invitrogen). DNA was counterstained with DAPI (1 µg/ml; Sigma-Aldrich) before coverslips were mounted in 90% glycerol + 10% Tris, pH 8.5, + 0.5% N-propylgallate on glass slides. Images were acquired on an AxioImager Z1 (100×, Plan Apochromatic oil DIC objective lens, 1.4 NA; all from Carl Zeiss) equipped with a charge-coupled device (CCD) camera (ORCA-R2; Hamamatsu Photonics) using the Zen software (Carl Zeiss) and blind deconvolved using Autoquant X (Media Cybernetics). Images were processed (contrast adjustments) in Adobe Photoshop CS4 (Adobe) and represent either maximum intensity projections of deconvolved z stacks (step size: 0.22 µm; DAPI; α-tubulin) or a single slice (CD8-GFP; lamin Dm0).
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