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sf9 cell culture protocol

Allow cells to attach for 15 minutes at room temperature in the hood. Suspension Cell Culture Sf9 Max Density >15 x106/mL Split Density 6-8 x 106/mL Seed Density 0.75-1 x 106/mL Split Frequency 2-3x/week It is recommended to passage the cells three days a week on a Mon/Wed/Fri schedule or twice a week on a Mon/Thurs or Tues/Fri schedule. The fed-batch method uses either a low-shear marine blade For screening of libraries and ELISA assays the b-βγ is stored in the elution buffer. a) Freezing medium: 45% conditioned (used or spent) medium, 45% fresh medium, 10% DMSO. Cell pellets are frozen in liquid nitrogen and stored at −80° until purification. 2) Centrifuge cells at 600xg for 5 minutes. Cells are cultured in suspension in Bellco glass conical flasks at 27°. Proceed to step 2. b. endstream endobj 3 0 obj <> endobj 6 0 obj <>/Font<>/ProcSet[/PDF/Text/ImageC]/Properties<>/XObject<>>>/TrimBox[0.0 0.0 595.276 793.701]/Type/Page>> endobj 7 0 obj <>/ExtGState<>/Font<>/ProcSet[/PDF/Text/ImageC]/Properties<>/XObject<>>>/TrimBox[0.0 0.0 595.276 793.701]/Type/Page>> endobj 8 0 obj <>/ExtGState<>/Font<>/ProcSet[/PDF/Text]/Properties<>>>/TrimBox[0.0 0.0 595.276 793.701]/Type/Page>> endobj 9 0 obj <>/Font<>/ProcSet[/PDF/Text]/Properties<>>>/TrimBox[0.0 0.0 595.276 793.701]/Type/Page>> endobj 36 0 obj <>stream uuid:cc3fad38-09e9-4b49-85b5-53aee592d436 Heterotrimeric His6-α1β1γ2 is eluted from the column with six successive 2 ml aliquots of 20 mM HEPES, pH 8.0, 100 mM NaCl, 0.1% C12E10, 10 μM GDP, and 150 mM imidazole. / The cells are infected at a density of 2–3 × 106 cells/ml with 7.5 ml of His6, αi1, 5 ml β1, and 2.5 ml γ2 viruses at approximately 1 × 108 pfu (plague-forming units)/ml each. To test the viability of the βγ in other assays the biotinylated βγ may have to be exchanged into another buffer. Culture parameters are monitored using a Beckman Coulter Vi-CELL TM XR and the cells are harvested 24 hours post infection. xmp.id:E5D95911C0E0E511B6A0DC672ADFAFDF Doubling time was approximately 48–72 h. Cells were passaged in a sterile cell culture hood using large-volume electric pipettors when they reached a concentration of 2–2.5 × 106 cells/ml into fresh TNM-FH media by dilution. xmp.iid:E4D95911C0E0E511B6A0DC672ADFAFDF ... (ZHAW), Switzerland, recently developed working protocols for the cultivation of Sf9 cells at high cell densities in SpodOmics. Shihori Tanabe, ... Tohru Kozasa, in Methods in Enzymology, 2004. These cells plus BacVector ® Insect Cell Medium are recommended for cotransfection of transfer plasmids with BacVector Triple Cut Virus DNA or BacMagic™ DNA for the construction of baculovirus recombinants and for transfection of pIEx™ … Keywords: Isotope labeling, Baculovirus, Nuclear magnetic resonance, Recombinant protein expression, Insect cells. Gibco® Sf9 cells (in Sf-900™ III SFM) were prepared as serum-free, suspension cultures from Sf9 cells that originated at the USDA Insect Pathology Laboratory. 2 Novagen • Insect Cell Expression Insect Cell Expression Vector Selection Guide Vectors Vector Size Cat. If the cells are removed from storage at − 70° they should be suspended in room temperature lysis buffer to facilitate thawing of the cell pellet. Cell culture and treatment. The supernatant is discarded and the cell pellets either frozen in liquid N2 and stored at − 70° for later use, or suspended for immediate processing in 15 ml of 4° lysis buffer [50 mM HEPES, pH 8.0, 0.1 mM EDTA, 100 mM NaCl, 10 mM 2-mercaptoethanol, 10 μM GDP, and 100 μg/ml phenylmethylsulfonyl fluoride (PMSF)]. Fractions containing the largest amount of βγ are used for the biotinylation reaction. 1 Cells are grown overnight in a shaking incubator at 130 rpm and 27°. False converted Imaging protocol is the same for Sf9 cells and for Aplysiasensory neurons except for the following: Sf9 cells are imaged with a x63 oil immersion objective whereas Aplysiasensory neurons are imaged with a x40 oil immersion objective. Muscarinic receptor yield is determined by [3H]NMS binding as described before. Sf9 cells are cultured in IPL-41 medium (JRH)/10% heat-inactivated fetal bovine serum (treated at 57° for 30 min)/0.1% (v/v) Pluronic F-68 (Invitrogen)/25 μg/ml gentamicin at 27° with constant shaking (140 rpm). The insect cells from the 800 ml culture are harvested by centrifugation in 500 ml culture bottles in a Beckman JA 10 rotor at 2400 rpm. 1.24.9 Insect Cell Culture. • High FiveŽ cells double every 18-24 hours. Usually 1–2 liters of culture at densities between 1 and 1.5 million cells/ml are infected with baculovirus. The serum-free Master Cell Banks were prepared at passage 26. Doubling time was approximately 48–72 h. • Recombinant protein expression from a variety of expression platforms The concentrations of wild-type and alanine-substituted His6–α-transducin proteins are determined by spectrophotometric means, using bovine serum albumin as a standard, and then normalized by the addition of storage buffer. ... on the surface area of the cell culture vessel (please refer to Table 1 on Page 2). Description; Overview: Sf9 Insect Cells are provided as frozen stocks of Spodoptera frugiperda Sf9 cells for establishment of cultures suitable for any application. Modification and secretion of … Naoyuki Iida, Tohru Kozasa, in Methods in Enzymology, 2004. No. The mixture is poured through a column and washed with 20 ml of dilution buffer. The reaction is allowed to proceed for 30 min at room temperature followed by addition of 10 mM ethanolamine pH 8.0 from a 200 mM stock and incubation on ice for 10 min. The ratio of virus to cell is 3:1. Fusion Tags Protease Cleavage Sites Insect Cell Expression N-terminal C-terminal ... • Sf9 Insect Cells and BacVector Insect Cell Medium (pages 14 ,15) customer .service@merckbio .com technical .service@merckbio .com Visit our website www … Seed 35 mm petri dishes with 2 ml of culture (1 x 10 6 cells). superSf9 insect cells can easily be cultured as monolayer cultures in T-flasks, suspension cultures in shake flasks, or scaled up for use in bioreactors. Cell Culture Protocol 6: Cell Counting Using a Hemocytometer. The column is warmed to room temperature for 15 min and washed with 5 ml of 30° wash buffer, 20 mM HEPES, pH 8.0, 100 mM NaCl, 10 μM GDP, and 1% cholate. Resuspended membranes carry the functional receptor. Transient plasmid DNA transfection protocol per well of a 6-well plate A. P0 baculovirus was shown to be most productive when Sf9 cells at transfection harvest have a viable cell count of 0.6–0.9 × 10 6 cells /ml, viability of 65% - 90%, and diameter of 25–26 μm compared to 0.8, > 91% and 21 μm for control cells, cell count being the most relevant parameter. In monolayer cultures, areas of infection display decreased density as cells die and lyse. This application report presents a simple protocol for achieving high-density culture of Spodoptera frugiperda (Sf9) cells using a New Brunswick bench-top, autoclavable stirred-tank reactor with a spin-filter assembly. This translates to 60–120 μg of receptor protein per liter of culture. The BIIC … Cells are lysed with a Potter–Elvehjem homogenizer, and centrifuged at 750 g for 10 min to remove intact cells and nuclei. Smith GE, et al. Sf9 cells are regular in size, easy to manipulate, and form good monolayers for plaque assays. Protocol for MIR 6100, 6104, 6105, 6106 ... developed for high-performance transfection of plasmid DNA into insect cells such as: Sf9, High Five™ (BTI-TN-5B1-4), and Drosophila melanogaster Schneider’s 2 (S2 or D. Mel. BIIC production begins by infecting a Sf9 insect cell culture at a multiplicity of infection (MOI) of 3 using recombinant baculovirus. Cell density doubles in approximately 24 h, and this growth rate should be monitored regularly. Sf9 cells (1 liter, 1.5 × 106 cells/ml) are infected with a recombinant baculovirus encoding rabbit PKCα (Fujise et al., 1994). After cell lysis (nitrogen cavitation at 500 psi for 30 min) and centrifugation (100,000g for 30 min), PKCα is purified chromatographically using DEAE-Sephacel, hydroxyapatite, phenyl-Superose HR10/10, and Mono Q HR5/5. Sf9 (Spodoptera frugiperda ovary) insect cells (Pharmingen, San Diego, CA) are grown in suspension in a shaking incubator (Innova 4000, New Brunswick Scientific, Edison, NJ) in IPL-41 medium (Gibco-Life Technologies, Rockville, MD) supplemented with 10% fetal bovine serum, 0.1% Pluronic F68 (Gibco-Life Technologies), gentamicin (50 μg/ml) and Fungizone (50 μg/ml) (Fungizone and gentamicin were from Gibco or the Tissue Culture Center, Washington University). Copyright © 2020 Elsevier B.V. or its licensors or contributors. Sf9 Culture and Membrane Preparation. Next, based on these parameters, the optimal ratio of DNA:PEI was tested using a fixed amount of bacmid … default Factors such as substrate concentration and metabolite buildup can be limiting for culture growth and viability at high densities. Protocols. For testing purposes, we used a culture of TriEx ™ Sf9 cells in serum-free TriEx Insect Cell Medium infected with a baculovirus recombinant expressing His•Tag ® β-galactosidase fusion protein, and compared the Insect PopCulture ® method with a conventional extraction and purification protocol that uses centrifugation to collect the cells and clear the lysate. Sf9 (Spodoptera frugiperda, GIBCO-BRL, Gaithersburg, MD) insect cells adapted to serum-free suspension culture are grown in Sf-900 II SFM (GIBCO-BRL). Sf9 cells are grown up from stock culture to 250 ml (0.5 × 106/ml) in IPL-41 medium containing 10% fetal bovine serum in two 250-ml flasks or a 500-ml flask. Plate cells 1. Cells are cultured in nonbaffled (Sf9 cells) or baffled (TN5 cells) shaker flasks at 150 rpm and 28°C. We recommend Sf9 or Sf21 cells for transfection, purification, and amplification of recombinant virus. The latter is resuspended in lysis buffer and spun down again (200,000g) to 45 min at 4°. Aim. Biologicals 22: 205-213, 1994. The resin is washed four times with Ni-NTA wash buffer (10 ml) at 25°. Sf9 and Sf21 cells can also be used for expression of recombinant proteins, but the High Five ™ cell line may produce higher levels. Cell densities are maintained between 0.5 and 3 × 106 cells/ml. Cell line selection . Centrifuge cells in Ti45 tubes (70mL) at … Suspension cultures of ≥ 95% viable cells were generated by seeding Sf9 cells into spinner flasks at 106 cells/ml at 27–28 °C and at 80–90 rpm without CO2 in the dark. Using a consistent number of cells will maintain optimum growth and also help to standardize … PDF/X-1a:2001 Shelley B. Hooks, T. Kendall Harden, in Methods in Enzymology, 2004. Samples are analyzed by silver staining14 and/or immunoblotting15 with the TF15 monoclonal antibody after electrophoresis through a 12% (w/v) nongradient sodium dodecyl sulfate (SDS)–polyacrylamide gel. The supernatant is diluted 5-fold with 20 mM HEPES, pH 8.0, 100 mM NaCl, 0.5% polyoxyethylene 10 lauryl ether (C12E10), 1 mM MgCl2, 10 mM 2-mercaptoethanol, 10 μM GDP, 100 μg/ml PMSF, and loaded onto a 2 ml Ni-NTA agarose column at 0.5 ml/min overnight. 4) Prepare freezing media and place on ice for > 15 min. The column is washed with 80 ml of 20 mM HEPES, pH 8.0, 1 mM MgCl2, 10 mM 2-mercaptoethanol, 10 μM GDP, 300 mM NaCl, 5 mM imidazole, 0.5% C12E10, and 100 μg/ml PMSF. PubMed: 7811453. The protein is eluted with 1 ml of elution buffer [50 mM NaPO4 (pH 8.0), 300 mM NaCl, 150 mM imidazole] and dialyzed against storage buffer [10 mM Na–HEPES (pH 7.6), 2 mM MgCl2, 1 mM DTT, 50% (v/v) glycerin]. See Subculturing Cellsto maintain and subculture Sf9 cells in suspension or adherent culture. Infected cells are harvested 48–60 hr later and resuspended in 50–100 ml of lysis buffer (50 mM HEPES, pH 7, 100 mM NaCl plus a cocktail of protease inhibitors : 1 mM benzamidin, 0.2 mM phenylmethylsulfonyl fluoride, and 10 μg/ml each of antipapain, leupeptin, aprotinin—the first two being added fresh). According to the results obtained at the above research institute, the cell … Adobe InDesign CS6 (Windows) PDF/X-1:2001 3. The supernatant is discarded and the membranes are suspended in 60 ml of extraction buffer, 50 mM HEPES, pH 8.0, 3 mM MgCl2, 50 mM NaCl, 10 mM 2-mercaptoethanol, 10 μM GDP and 100 μg/ml PMSF, using a Dounce homogenizer. H��W�n9}��c7��;{��V2ޙ�I2��Ef0P���I���~�_�S��ڒ�V�ua��Xs�����8��P��]�^`�(������m���bV��f���]B�Q���`���*�f��c���+eK��ܴ�r����.�b��~��TLj^+�f�>�̰ �3�:����MTw>�&B��j�Ӡ��$J>'�����2'��&h(��V�?������[�؜m+�i�W��6~.�m��/��|��m����ݮ��*��,�oZ6��n��n��]ܮ6=>�7����9go�X��'^K�R��nK]s����y����Dp�fKHcC�g�\����e�]��{���m�gۻ~�_-��V)fՉ�'a��W��]�6?t�o�Sk�)�. SpodOmics™ is the name of our chemically defined medium for the in vitro cultivation of Spodoptera frugiperda (Sf9) cells under protein- and peptide-free culture conditions. 2016-03-02T16:45:33-05:00 Note:If Sf9 cells are thawed from frozen stock, allow at least two weeks for the cells to recover and enter the exponential phase of growth. Culture mode Sf9 insect cells can be grown using any of the three common culture methods, batch, fed-batch, or continuous, but they are usually grown using fed-batch or continuous culture because of the increased yields. ECACC Laboratory Handbook 4th Edition. Biotinylated β1γ2 subunits are prepared using a modification of the method developed by Dingus et al.9 Sf9 (Spodoptera frugiperda ovary) insect cells grown in suspension are infected with His6-αi1, β1, and γ2 with modifications to what has been previously described by Kozasa and Gilman.10 The sf9 cells are grown in 800 ml Sf 900 II medium (Gibco-BRL, Gaithersburg, MD) in a 2 liter culture flask with shaking at 125 rpm at 27–28°. The former media is much better for Sf9 cells in adherent cultures for recovering them from frozen stocks. An increase in average cell diameter of 10% or greater 24 hours post infection ensures that the insect cells were properly infected. Inaki Azpiazu, N. Gautam, in Methods in Enzymology, 2002. Cell cycle progression was studied in serum-free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. PDF/X-1:2001 Plate 8 × 10 5 Sf9 or Sf21 cells per well. Infected cells were harvested by centrifugation at 3500× g for 10 min at 4 °C. We obtain 1–2 nmol [3H]NMS binding sites per liter of cell culture. bly, production, and expression of recombinant gene product. TN5 insect cells (comparable to High Five cells, Invitrogen, Carlsbad, CA) adapted to serum-free suspension are grown in EX-CELL 401 (JRH, Lenexa, KS) supplemented with 2 mM L-glutamine and 100 U/ml penicillin/streptomycin. 2016-03-02T16:45:34-05:00 from application/x-indesign to application/pdf It is not advised to repeatedly allow the cells to reach maximum density as the growth kinetics of the culture may change. Adobe InDesign CS6 (Windows) 2016-03-02T16:45:33-05:00 Two ml of washed Ni-NTA agarose is added and incubated with mixing overnight at 4°. On the day before an infection, cells at a density of approximately 3 × 106 cells/ml are diluted to 0.8 × 106 cells/ml in Grace's medium without FBS, resulting in a final concentration of FBS of approximately 3%. cells per mL in a non-humidified, non-gas-regulated environment. Large Scale Preparation of Membrane Containing Over-expressed Proteins from SF9 Cells December, 2008 Version 1.0 Summary: This protocol is currently being used at the JCIMPT to prepare samples of membranes . For the majority of manipulations using cell lines, such as transfections, cell fusion techniques, cryopreservation and subculture routines it is necessary to quantify the number of cells prior to use. The medium carried over to the next step will enhance the transfection efficiency. The yield is 1 mg from a 1-liter culture, the specific activity is 800 units/mg, and the protein is more than 90% pure based on silver staining after gel electrophoresis. Suspend 5L of cell culture in 400mL of lysis buffer b. 3) Remove the media and save. Culture Sf9 cells in an orbital shaker incubator at 135 rpm and 28 °C in a polycarbonate Erlenmeyer flask containing serum-free insect culture medium1,2,3. The suspension is frozen and thawed 4 × by alternately plunging the 50 ml tube containing the suspension into liquid N2 and thawing in a 37° water bath. Adobe PDF Library 10.0.1 Most insect cells can be cultivated over a temperature range of 25–30 °C, but the optimal temperature for cell growth and infection for Sf21 and Sf9 cells is considered to be 27–28 °C, when they will double every 18–22 h. NHS-LC-biotin (Pierce, Rockford, II) is added from a 20 mM stock in dimethyl sulfoxide (DMSO) to give a final concentration of 1 mM. For the ELISA we have successfully used both immobilization via biotin or immobilization of the βγ directly to the plastic well of the dish. Cell densities and viabilities were monitored daily throughout culture lifespan. 256_BioFloCelliGen115_Sf9_Rev2.indd The surfactant Pluronic F-68 (Gibco BRL) is added to the culture medium to a final concentration of 0.1% to protect cells from lysis. Volume of media was kept at 30–50% of flask volume to optimize aeration and shear stresses. Proteins are extracted from the membrane fraction by addition of cholate to 1% to the suspended membrane fraction with slow stirring at 4° for 1 h. Detergent-insoluble particulate matter is removed by centrifugation at 100,000 g for 45 min at 4°. The eluted fractions are assayed for protein using an amido black protein assay11 and the fractions are separated on a 12% sodium dodecyl sulfate (SDS)–polyacrylamide gel and stained with Coomassie blue. The lysate is then diluted to 100 ml with lysis buffer and the particulate fraction containing membranes is harvested by centrifugation at 100,000 g for 45 min at 4°. Sf9 cells were cultured in TNM-FH medium (Trichopulsia ni medium—Formulation Hink), consisting of Supplemented Grace’s Insect Cell Medium, supplemented with the addition of 10% FBS-HI (American origin, Invitrogen), 1 × penicillin–streptomycin (Invitrogen), and 10 μg/ml gentamicin (Sigma). ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. URL: https://www.sciencedirect.com/science/article/pii/S007668790129106X, URL: https://www.sciencedirect.com/science/article/pii/B9780124080515000103, URL: https://www.sciencedirect.com/science/article/pii/S0076687904900183, URL: https://www.sciencedirect.com/science/article/pii/S0076687904900298, URL: https://www.sciencedirect.com/science/article/pii/S0076687904900110, URL: https://www.sciencedirect.com/science/article/pii/B9780124200708000131, URL: https://www.sciencedirect.com/science/article/pii/S0076687901291083, URL: https://www.sciencedirect.com/science/article/pii/S0076687902450057, URL: https://www.sciencedirect.com/science/article/pii/S007668790244709X, URL: https://www.sciencedirect.com/science/article/pii/S0076687902447404, Regulators and Effectors of Small GTPases, Regulators of G-Protein Signaling, Part B, Laboratory Methods in Enzymology: Protein Part A, Also known as Grace's insect cell medium-supplemented, Elena Smirnova, ... Alexander M. van der Bliek, in, Kosei Moriyama, ... Robert F. Margolskee, in, G Protein Pathways, Part B: G Proteins and their Regulators. %PDF-1.3 %���� 2016-03-02T16:45:34-05:00 Biotinylation is confirmed by electrophoresis and blotting with streptavidin–horseradial peroxidase (HRP) and by showing that all of the biotinylated βγ could be bound to streptavidin agarose. containing over-expressed proteins from biomass of SF9 cells. ... Culture cells in the appropriate medium, with or without serum. Stock cultures (50 ml) of cells in a 100-ml flask are subcultured every 3 days with a density between 0.5 and 4 × 106 cells/ml. The infection is begun the following day at a cell density of approximately 1.5 × 106 cells/ml. Remove medium from dishes and replace with 100 µl virus dilution. Expand Sf9 cultures by seeding shake flasks at 3 to 5 x 105viable cells/mL by diluting cells in pre-warmed growth medium. David A. Healthy Doubling Times: • Sf9 cells double every 72 hours • Sf21 cells double every 24 hours. Transient plasmid DNA transfection protocol per well of a 6-well plate A. After 2 or 3 days, cells are transferred to four 2-liter flasks containing 750 ml of scale-up medium [IPL-41/1% fetal bovine serum/1% chemically defined lipid concentrate (Invitrogen)/0.1% (v/v) Pluronic F-68/25 μg/ml gentamicin]. Production of the BTV-2 recVP7 was scaled-up to 1.5 L of Sf9 cell culture. protocol for this technique can be found in Adherent Cell Culture, page 13. This modification results in a higher titer of virus, which is useful for efficient protein expression. The cells are suspended in PBS (10 mM KPO4, pH 7.4, 150 mM NaCl) and transferred to a 50 ml conical culture tube and centrifuged at 3000 g for 30 min at 4°. xmp.did:03801174072068118C14E45E7B4C0E64 After 2 or 3 days, they are further expanded to 1 liter (about 0.5–1 × 106/ml). 5) Resuspend the cells in the cold medium and aliquot 1 ml of cells into 2ml cryo-vials. The Sf9 insect cell line is a clonal isolate derived from the parental Spodoptera frugiperda cell line IPLB-Sf-21-AE, and it is a suitable host for expression of recombinant proteins from baculovirus expression systems (e.g., Invitrogen’s Bac-to-Bac® and Bac-N-Blue™ Expression Systems). Sf9 (Spodoptera frugiperda ovary) insect cells are grown in a 0.5-liter round-bottom spinner flask with 0.25 liter complete medium [Grace's insect medium with supplements (GIBCO-BRL, Grand Island, NY) and 10% fetal bovine serum (FBS)] at 27°. Prepare a 100–200 mL culture of Sf9 cells at an appropriate cell density in serum-free SF900 II insect cell culture medium (e.g., 2 × 10 6 Sf9 cells/mL … The images were obtained using 10X and 20X objectives (panels A and B, respectively) 3 days after seeding . The typical yield from 1 liter of Sf9 cells varies form 50 to 100 μg of α-transducin protein. Protocols for Sf9 cell culture and baculovirus expression are available as manuals,9 or from commercial sources for baculovirus vectors (e.g., Gibco-Life Technologies, Pharmingen, San Diego, CA; Invitrogen, Carlsbad, CA).

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