S2 cells were cultured in 25-cm2 flasks using Shields and Sang M3 insect media (Sigma-Aldrich). For transfection, when cells were ∼90% confluent, the medium was removed and 6 ml of fresh M3 medium was added to the flask. Cells were gently resuspended by pipetting and added to a 12-well plate with 1 ml of cells per well. After 2 h, once the cells had adhered to the bottom of the well, the M3 media was replaced with 1 ml serum-free M3 medium, and the cells were transfected with 1 µg of each DNA using Lipofectamine 2000 following the manufacturer’s protocol. After 16 h, the serum-free medium was replaced with 1 ml M3 medium containing serum. For experiments with furin RNAi, 5 µg of dsRNA was used for transfection. Under all conditions, transient expression was examined 2–3 d after transfection.
Shields and sang m3 insect media
Manufactured by Merck Group
Sourced in United States
Shields and Sang M3 insect media is a laboratory product used for the cultivation of insect cell lines. It provides a defined, serum-free formulation to support the growth and maintenance of various insect cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Minimal essential medium, by Corning (2 mentions)
Penicillin streptomycin antimycotic, by Corning (2 mentions)
L glutamine, by Corning (2 mentions)
Fetal bovine serum (fbs), by Corning (2 mentions)
Non essential amino acids (neaa), by Corning (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Insect mediasupplement, by Merck Group (1 mentions)
900 airyscan, by Zeiss (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Effectene transfection reagent, by Qiagen (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Schneider s insect media, by Merck Group (1 mentions)
7 protocols using shields and sang m3 insect media
1
Transient Transfection of S2 Cells
2
Schneider 2 Cell Maintenance and Transfection
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Schneider 2 cells were grown and maintained in Shields and Sang M3 insect Media
(Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% insect media
supplement (Sigma-Aldrich) with appropriate antibiotics at 25 °C. Cells
were subcultured every five days at a 1:5 ratio when confluency reached around
100%. The DDAB method of transfection was employed with some modifications for
24-well plates (Han, 1996 (link)). Cells were
collected and assayed three days after transfection.
(Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% insect media
supplement (Sigma-Aldrich) with appropriate antibiotics at 25 °C. Cells
were subcultured every five days at a 1:5 ratio when confluency reached around
100%. The DDAB method of transfection was employed with some modifications for
24-well plates (Han, 1996 (link)). Cells were
collected and assayed three days after transfection.
3
Mitochondrial Membrane Potential in Fly Gut
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Three--day-old flies (15 females and 10 males) were transferred in vials with semi-defined media with or without 1 mM sodium phosphonoformate tribasic hexahydrate (PFA) (Sigma, P6801). After 2 wk, fly guts (midgut, R4-R5 section) from both control and PFA-treated groups were dissected in Shields and Sang M3 Insect Media (Sigma, S8398) with or without 1 mM PFA added. Dissected guts were stained with 1 μM TMRE (Sigma, 87917) in the same media condition for 20 min at room temperature. After two washes with the corresponding media supplemented with 1 μM Hoechst 33342 (Thermo Scientific, 62249) and 0.01 μM TMRE, live imaging of the gut was performed on Zeiss 900 Airyscan. Intensity of TMRE was quantified in Fiji, and background was subtracted. Figures were prepared in Prism v9.
4
Live-cell imaging of BG-2 cell migration
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DM-BG2 cells (referred to as BG-2 cells in this paper) are cultured in Shields and Sang M3 insect media (Sigma), 10% fetal bovine serum (Serum), 10 μg ml−1 insulin (Sigma), 1% penicillin–streptomycin (Gibco) at room temperature. For experiments to validate the Bayesian inference model, brightfield images are acquired every 5 min for 180 min on a Nikon Ti microscope. Electronic supplementary material, movie S3 is a representative movie where transition frequency is analysed. For other experiments, cells are transfected with plasmids encoding actin-GAL4 and UAS-EGFP using Effectene transfection reagent (Qiagen). In the electronic supplementary material, figures S1 and S3a, and movie S3, we use a plastic pipet tip to remove cells from a region of the plate and thus create a region of free space for BG-2 to migrate into.
5
Culturing Insect and Mammalian Cells with Wolbachia
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Drosophila melanogaster cells (JW18) with and without Wolbachia (strain wMel) and Aedes albopictus cells (C710) with and without Wolbachia (strain wStri) were grown at 24 °C in Shields and Sang M3 insect media (Sigma-Aldrich, Burlington, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Waltham, MA, USA), 1% each of l -glutamine (Corning, Corning, NY, USA), non-essential amino acids (Corning, Corning, NY, USA) and penicillin-streptomycin-antimycotic (Corning, Corning, NY, USA). Baby hamster kidney fibroblast (BHK-21) cells were grown at 37 °C under 5% CO2 in 1× Minimal Essential Medium (Corning, Corning, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (Corning, Corning, NY, USA), 1% each of l-glutamine (Corning, Corning, NY, USA), non-essential amino acids (Corning, Corning, NY, USA) and penicillin-streptomycin-antimycotic (Corning, Corning, NY, USA).
6
Drosophila S2 Cell Culture and Virus Infection
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Drosophila S2 cells were maintained in Shields and Sang M3 insect media (Sigma-Aldrich, Oakville, ON, Canada) supplemented with 10% fetal bovine serum (SSM3) at 25 °C as described previously [7 (link)]. For virus infections, S2 cells were washed with PBS, then incubated with CrPV for the indicated MOI for 30 min and subsequently, washed with PBS and resuspended with SSM3. Viral titers were determined by a fluorescence-focusing assay as described [7 (link)].
7
Culturing Wolbachia and Insect/Mammalian Cells
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Wolbachia (a generous gift from Dr. Horacio Frydman, Boston University) were grown in 1X Minimal Essential Medium (Corning) supplemented with 5% heat-inactivated fetal bovine serum (Corning), 1% each of L-Glutamine (Corning), non-essential amino acids (Corning) and penicillin-streptomycin-antimycotic (Corning). Aa23 Aedes albopictus cells with and without Wolbachia wAlbB (a generous gift from Dr. Horacio Frydman, Boston University) were grown at 24 ºC in Schneider's insect media (Sigma-Aldrich) supplemented with 20% heat-inactivated fetal bovine serum (Corning), 1% each of L-Glutamine (Corning), non-essential amino acids (Corning) and penicillin-streptomycin-antimycotic (Corning). JW18 Drosophila melanogaster cells with and without Wolbachia wMel were grown at 24 ºC in Shields and Sang M3 insect media (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum, 1% each of L-Glutamine (Corning), non-essential amino acids (Corning) and penicillin-streptomycin-antimycotic (Corning). Aedes albopictus C636 cells and mammalian BHK-21 and Vero cells were grown at either 28ºC (C6/36) or 37 ºC (BHK/Vero) under 5% CO2 in 1X Minimal Essential Medium (Corning) supplemented with 10% heat-inactivated fetal bovine serum (Corning), 1% each of L-Glutamine (Corning), non-essential amino acids (Corning) and penicillinstreptomycin-antimycotic (Corning).
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