A cell sorting protocol for selecting high-producing sub-populations of Sf9 and High Five™ cells. This kit is a second-generation method that replaced our earlier Sf9 Insect Cell HCP ELISA Kit (F020). USA 82: 8404-8408, 1985. The protocol is carried out to prepare a membrane homogenate prior to membrane extraction and detergent solubilization. While cells are attaching, make 1:10 serial dilutions of virus in media + 10% FBS (the range I usually do is 10-1 to 10 … ESF-921 insect cell culture medium (Expression Systems, 96-001-01). The typical yield from 1 liter of Sf9 cells varies form 50 to 100 μg of α-transducin protein. • High Five™cells double every 18–24 hours. 1. Summary. ���� K�N*��iDzR;��Iǥ�N_�T�V��`�������X�-�`4p� � Andrew B. Kleist, ... Brian F. Volkman, in Methods in Cell Biology, 2019. Insect Cells Introduction to Insect Cell Analysis Although there are many insect cells that are routinely studied and used in research, the most common is the Sf9 cell line. This kit is a second-generation method that replaced our earlier Sf9 Insect Cell HCP ELISA Kit (F020). PERT Assay Protocol. 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 . x�}�Oh�Pǿ���t2p�1T���C;��2/��mu�����di�ƥILҪcģsG�T���$��oE'��E�CaP����ɰ��������{��h�D��B. See attached document for all growth and cell characteristics. April 6th, 2011 • Carole A. Farah 1, Wayne S. Sossin 1. Split cells 1 confluent 100mm plate into 1 150mm plate. Early Phase: Infection begins with the adsorptive endocytosis of one or more competent virions by a cell in a high metabolic state (peak replication rate). 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. The cells are prepared from low passage cells (<30 passages in ESF 921™ medium) and supplied in a cryogenic vial containing 1×10 7 cells. ‘Complete’ TNM-FH insect cell medium (for Sf9 cell culture), ‘Complete’ 2× Grace's insect cell medium (for plaque overlays), Add water to dissolve salts, adjust to pH 7.4 with HCl, adjust final volume to 500 ml, Extraction Buffer 1 (mild protein extraction conditions), Add water to dissolve salts, adjust to pH 8.0 with HCl, adjust final volume to 500 ml, Extraction Buffer 2 (harsh protein extraction conditions), Elena Smirnova, ... Alexander M. van der Bliek, in Methods in Enzymology, 2001. Important Guidelines for Transfection Cellfectin® II is a lipid suspension that may settle with time. Usually 1–2 liters of culture at densities between 1 and 1.5 million cells/ml are infected with baculovirus. Sf9 cells are adapted for suspension culture in ESF 921 or ESF AF and are available as a frozen vial or suspension culture.Sf9 cells are commonly used to produce recombinant baculoviral stocks and to produce recombinant proteins. 5. As an alternative to the spinner flask of producing high-titer virus stock described in the manual, we also use cells growing adherently in 10 tissue culture flasks of 175 cm2 each containing 20 × 106 cells in 30 ml medium. Use 1 × 10 6 cells/dish for Sf9 cells and 1.5 × 10 6 cells/dish for Sf21 cells in 2 mL of SF900 II insect cell culture medium. The number of cells per square x 10 4 = the number of cells/ml of suspension. The following . �m �#$��! They can be grown in the absence of … << /Length 8 0 R /N 3 /Alternate /DeviceRGB /Filter /FlateDecode >> Figure 1 Vertiga Shaker manufactured by Thomson Instruments Co. Summary: This protocol is used to generate baculoviruses containing GPCR genes of interest for expression in SF9 cells. Here, we provide simplifi … Infect 20 150mm plates of SF9 cells with 100ul of virus stock. Continue to maintain the cells in culture until the viability of the recovered cells is confirmed (see Step 9). ���"Y2}QX=`8�X��n_e���j�q���fp�Q��f��m\��&�����頰�o�j9�Jj��J�����f��u&I���B�G�2�mt�B�dR�uש"��s;ix������}�P���DV�>��8�GS�:8=��L{��+[p��5�.��?�=��E-�z(��� 5-q����Gf ��r�{��1�PP��U�&�E��:~d��yR�K�9��ڱ����,� Sf9 Insect Cells. Sf9 cells are adapted for suspension culture in ESF 921 or ESF AF and are available as a frozen vial or suspension culture.Sf9 cells are commonly used to produce recombinant baculoviral stocks and to produce recombinant proteins. (2)). The Cell Line Optimization Protocol enables you to optimize 4D-Nucleofection™ Conditions for a cell line of your choice using our Cell Line Optimization 4D-Nucleofector X Kit. Cells are harvested 48 h after infection by centrifugation at 2000 rpm at 4° for 15 min in a JLA 10,500 rotor (Beckman). Susceptibility of the Sf9 insect cell line to infection with adventitious viruses. Nuclei and cells are spun down at 500g for 10 min at 4° and the supernatant is spun down at 200,000g which results in a compact microsomal pellet. growth rate, metabolite production and consumption rates) will be measured to refine and validate the metabolic reconstruction Bernal, Vicente, Nuno Carinhas, et al. We obtain 1–2 nmol [3H]NMS binding sites per liter of cell culture. Retroviral vectors have become an indispensable tool in the modern molecular biology laboratory. Cells are harvested 48 h after infection and suspended in 120 ml of ice-cold lysis buffer [20 mM Tris–HCl (pH 7.5), 5 mM EGTA, 1 mM EDTA, 10 mM 2-mercaptoethanol, and protease inhibitors]. The Sf9 insect cell line is a clonal isolate derived from the parental Spodoptera frugiperda cell line IPLB-Sf-21-AE. Dilute SF9 cells to a concentration of 5x10e5 cells/mL and seed 2mL well in 2 x 6 well dishes. l-Methionine (methyl-13C, 99%) (Cambridge Isotope Laboratories, CLM-206-1). Description; Overview: The unique TriEx™ Sf9 Cells are derived from a high-yielding clone of Sf9 cells. This is a phase I, single-center, randomized, placebo-controlled, double-blind study, to evaluate safety, tolerability and immunogenicity of a recombinant SARS-CoV-2 vaccine (Sf9 cell) in Chinese healthy population aged 18 years and older. Integral membrane proteins have a critical role in fundamental biological processes; they are major drug targets and therefore of high research interest. Acad. 2 0 obj For Sf21 cells: Plate cells at a density of 0.75 x 106 cells/ml. PubMed: 7811453. Here, we provide simplified protocols that facilitate the generation of high-quality virus and initial expression analysis for integral membrane protein targets utilizing the baculovirus-mediated expression system in insect cells. Healthy doubling times: • Sf9 cells double every 24–30 hours • Sf21 cells double every 24–30 hours. The cell lines are also suitable for use in the InsectSelectŽSystem. Ӽy_��gy7;4֙NlS�6�χ�E]�$5��{�q]�{ߑ}����y4��zN( ��"�� �V��?1X6��ҁ�C�T.KY�I>��-�����]V��ّ�2� X��лف1\Z�#@J�Š�k��A������Q��K`�g�ߦ/�� �~ �������.����D���(iA�ӥ�}Ä%]�O�;e1�W�@�7��'��рڶ��.���Y2�TB\Tp�$B1���B�6L��"O��P���ס��E���O���^`�m��ӰK}{�\��:�V�����?p+��P�� ��Y�����'��Dلlm�teB��G����&�\�j�.Lqh��ܦ[HO�|��-��Ћ�j�Pb�z]�M�76����N^`��&�磆< After randomization, the trial for each subject will last for approximately 13 months. The peak fraction is mixed with 400 μl of Ni-NTA resin (Qiagen, Valencia, CA), which is preequilibrated with Ni-NTA wash buffer [50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 20 mM imidazole], and then rotated for 1 hr at 4°. protocol in the lab). 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). Description; Overview: Sf9 Insect Cells are provided as frozen stocks of Spodoptera frugiperda Sf9 cells for establishment of cultures suitable for any application. Allow the cells to attach by incubating at 28°C for 20 min. This protocol is a slight modification of Expression Systems protocols for generating viruses. 3. The protocol for this technique can be found in “Adherent cell culture“ on page 18. Prepare cryopreservation medium as follows: 7.5% DMSO, 46.25% "ready to use" SFM, 46.25% conditioned SFM (medium used for growing Sf9 cells for 2-3 days, sterile filtered) as appropriate and store on ice.Harvest cells in mid log growth phase by centrifuging at 1000x g for 5 mins. For transfection of adherent cells, 2 ml Sf9 cells with 0.8 × 10 6 cells in Ex-Cell 420 supplemented with 5% FCS were seeded per well of a 6-well plate 1–5 h prior to transfection. Smith GE, et al. Sf9 cells are regular in size, easy to manipulate, and form good monolayers for plaque assays. General description Sf9 Insect Cells are provided as frozen stocks of Spodoptera frugiperda Sf9 cells for establishment of cultures suitable for any application. The protocol that came with the cells (Sf9) presented both the adherent and suspension cultures methods so I did not consider that any of them would give me trouble. Dounce cells 30 times, or sonicate for 2 minutes. Corning ® T-75 flasks (catalog #430641) are recommended for subculturing this product. General protocols for inducing apoptosis in cells Apoptosis may be induced in experimental systems through a variety of methods. Screening period is 1 week prior to randomization (Day -7 to Day -1), and each dose of either SARS-CoV-2 vaccine (Sf9 Cell) or placebo will be given intramuscularly (IM) on Day 0 and Day 28 for a two-dose regimen, or on Day 0, Day 14, and Day 28 for a three-dose regimen. Sf9 cells (1 liter, 1.5 × 106 cells/ml) are infected with a recombinant baculovirus encoding rabbit PKCα (Fujise et al., 1994). Transfection protocols for Sf9 and Sf21 cells are described in this manual. 11496-015. 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°. Cell viability was maintained at ≥ 95% viable cells as determined by Trypan blue exclusion (1 μl of 0.4% Trypan blue in phosphate-buffered saline (PBS) with 10 μl of cell suspension, measured on a hemacytometer). 1 0 obj A. The cells (usually 1.5–2 × 106/ml) are infected with amplified, recombinant baculoviruses the next day. Volumes used in this protocol are for 75 cm 2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Transfection of Sf9 cells in suspension December, 2008 Version 1.0. Sf9 Insect Cells. See page 2 to view a procedure for transfecting Sf9 or Sf21 with Baculovirus DNA. Dounce cells 30 times, or sonicate for 2 minutes. Shelley B. Hooks, T. Kendall Harden, in Methods in Enzymology, 2004. Summary In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. • Sf9 cells, as shown in Fig. 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%. endobj This modification results in a higher titer of virus, which is useful for efficient protein expression. They allow stable expression of a gene of interest in dividing cells and stable gene knock-down by expression of short hairpin RNA (shRNA). • Adapted to serum-free suspension growth in Sf-900 II SFM, a serum-free medium optimized for growth of Sf9 and other invertebrate cell lines5. Stable transfected Sf9 cells which emit eGFP upon infection with baculovirus. 2. Incubate cell cultures at room temperature for 1 hour. Routine growth of Sf9 cells in suspension culture : Freezing Sf9 cells for storage in liquid nitrogen. I was growing my SF9 cells with supplemented media and 10% FBS, antibiotics, and antimycotic. By continuing you agree to the use of cookies. For Sf9 cells: Plate cells at a density of 0.5 x 106 cells/ml. Inaki Azpiazu, N. Gautam, in Methods in Enzymology, 2002. Cell densities in excess of 8.3 x 10^6 cells/ml can be achieved with suspension cultures of Sf9 cells using Insect-XPRESS™ Medium and an excess of oxygen. After 2 or 3 days, they are further expanded to 1 liter (about 0.5–1 × 106/ml). Zhang PF, et al. Shihori Tanabe, ... Tohru Kozasa, in Methods in Enzymology, 2004. PROTOCOL FOR COUNTING CELLS: 1) Get SF9 cells from the incubator shaker, and put in TC hood 2) Remove 10 uL of the cells, being sure to shake the flask beforehand 3) Dilute the cells 5x with 40 uL of Trypan blue 4) Place 10 uL in a hemocytometer (from Shirley’s bench) 5) Count the cells in 4 square grids 1 grid = 10^-4 mL Cell Density = (mean # of cells per grid)*5*10^4 cells/mL. This application note presents a simple protocol for achieving high-density culture of Sf9 insect cells using an Eppendorf benchtop, autoclavable stirred-tank bioreactor. SF9 Insect Cell Host Cell Proteins 2nd Generation Immunoenzymetric Assay for the Measurement of SF9 Insect Cell Host Cell Proteins Catalog # F840 Intended Use This kit is intended for use in determining the presence of host cell protein contamination in products manufactured by expression in SF9 insect host cells. << /ProcSet [ /PDF /Text ] /ColorSpace << /Cs1 3 0 R >> /Font << /F1.0 The protocol is carried out to prepare a membrane homogenate prior to membrane extraction and detergent solubilization. Description; Overview: The unique TriEx™ Sf9 Cells are derived from a high-yielding clone of Sf9 cells. Resuspended membranes carry the functional receptor. B825-01) and Sf21 (Catalog no. Sf9 and Sf21 cells can also be used for expression of recombinant proteins, but the High FiveŽ cell line may produce higher levels. Protocol for: Madhi SA, Polack FP, Piedra PA, et al. Protocol for MIR 6100, 6104, 6105, 6106 Quick Reference Protocol, ... 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. DOI: 10.1016/j.jbiotec.2013.10.020 Corpus ID: 5476283. 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). A. Description; Overview: Sf9 Insect Cells are provided as frozen stocks of Spodoptera frugiperda Sf9 cells for establishment of cultures suitable for any application. Recombinant protein production is the first step in the protein tool generation for biochemical and biophysical studies. Kosei Moriyama, ... Robert F. Margolskee, in Methods in Enzymology, 2002. endobj Quickprep miniprep using an isolation filter may work, but DNA must be sterile. 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. TIPS protocol. 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. Prepare Supernatant Spin lysate at top speed (~18,000 rpm) for 1 hour *Take sample of supernatant for SDS PAGE gels 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). Plate cells in ___ml complete growth medium (per well). Cell densities are maintained between 0.5 and 3 × 106 cells/ml. endobj Transfection of Sf9 cells in suspension December, 2008 Version 1.0. 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). containing over-expressed proteins from biomass of SF9 cells. Cells are lysed with a Potter–Elvehjem homogenizer, and centrifuged at 750 g for 10 min to remove intact cells and nuclei. 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. c Single-step procedure presented in this study. A. Sf9 cells in 50 ml suspension culture at a density of 2 x 10 6 cells/ml were infected at a MOI of 2. Gibco® Sf9 cells are commonly used to isolate and propagate recombinant baculoviral stocks and to produce recombinant proteins. PubMed: 3878519 Red blood cells are typically too small and numerous for this protocol … We obtain serum from Atlanta Biologicals (Atlanta, GA). Used in titration assay to determine bacculovirus titer in lieu of CPE or Plaque assays. Originally derived from the Spodoptera frugiperda (fall armyworm) moth and adapted for cell culture. stream Lyse Cells Add ~20 ml lysis buffer/liter of SF9 cells harvested, sit on ice for 20 minutes. Remove and discard culture medium. Incubate cell cultures at room temperature for 1 hour. stream x��VMo�@�ﯘK��q��*���[m��Fj�l�b��UWM���$^��T>`�y3�͛�������O����o����b)��GE�jq�������yD| Transfections with pBlueBac constructs, determining the virus titer with a plague assay, purification of recombinant plagues, and generating a high-titer virus stocks are done as described in the MaxBac 2.0 Transfection and Expression Manual (Invitrogen, San Diego, CA). For Sf9 cells: Plate cells at a density of 0.5 x 106 cells/ml. Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons Article doi: 10.3791/2516. Glass culture flasks with steel closures of various sizes (10 ml to 2 liters) (Bellco) are used for suspension cultures. Equilibrate 2 ml solid beads/liter of SF9 cells by washing with lysis buffer. The Sf9 cell line is a clonal isolate of IPLBSF21-AE (Sf21). @article{Vidigal2013ACS, title={A cell sorting protocol for selecting high-producing sub-populations of Sf9 and High Five™ cells. ESF-921 Delta Series methionine-deficient insect cell culture medium (Expression Systems, 96-200). Determined conditions are transferable to the 100 µl single Nucleocuvette™. The Sf9 cell line is a clonal isolate of Sf21 and has become the cell line of choice in many laboratories for working with recombinant baculoviruses. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Volume of media was kept at 30–50% of flask volume to optimize aeration and shear stresses. endstream Dounce ~50 strokes on ice using the B pestal. Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector. This translates to 60–120 μg of receptor protein per liter of culture. cells/mL, determine viable and total cell counts (see procedure). Cell densities and viabilities were monitored daily throughout culture lifespan. During the 1-h incubation period, prepare a DNA–liposome complex cotransfection mix of BacMagic DNA and Insect GeneJuice ® transfection reagent for each transfection. Allow cells to recover for ~1 hr. 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). We use cookies to help provide and enhance our service and tailor content and ads. 4 0 obj For highest convenience the initial optimization step is performed in the 16-well Nucleocuvette™ Strip format. Sf9 insect cells can easily be cultured as monolayer cultures in T-flasks, suspension cultures in shake flasks, or scaled up for use in bioreactors. Plate cells approximately 1-2 hours prior to transfection 1. 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]. Donald L. Jarvis, in Methods in Enzymology, 2014, Baculovirus vectors [e.g., Autographa california nucleopolyhedrovirus (Ac NPV)], Step 1. ���'x���!�`��Y�1���pU�Lr�ZVI�D�N�|��?�8l2z^�`S Cellfectin®II Transfection Protocol Transfection protocols for Sf9 and Sf21 cells are described in this manual. The latter is resuspended in lysis buffer and spun down again (200,000g) to 45 min at 4°. Sf9 cells, a clonal isolate of Spodoptera frugiperda Sf21 cells (IPLB-Sf21-AE), are commonly used in insect cell culture for recombinant protein production using baculovirus.They were originally established from ovarian tissue. Incubate at 300C for 5 days. To obtain the best results, perform transfections using the appropriate protocol for your system and cell type. A. Sf9 cells in 50 ml suspension culture at a density of 2 x 10 6 cells/ml were infected at a MOI of 2. 500ml per 2L roller bottle. Biologicals 22: 205-213, 1994. Cell densities in excess of 8.3 x 10^6 cells/ml can be achieved with suspension cultures of Sf9 cells using Insect-XPRESS™ Medium and an excess of oxygen. [ 0 0 612 792 ] >> Population doubling times for insect cells will vary depending on growth conditions. Cells were initially cultured in monolayers in tissue culture treated T75 flasks (Corning) until 90% confluent (~ 107 cells) and passaged no more than 20 times. Impure preparations of plasmid DNA are toxic to the cells, and many cells may lyse shortly after transfection. 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. Muscarinic receptor yield is determined by [3H]NMS binding as described before. Gross, David L. Silver, in Methods in Cell Biology, 2013. containing over-expressed proteins from biomass of SF9 cells. Protocol This trial protocol has been provided by the authors to give readers additional information about their work. Roche cat. (2009). B821-01) cell lines are the traditional cell lines used with baculovirus and originated at the USDA Insect Pathology Laboratory. Sf21 cells are somewhat more disparate in size and form monolayers and plaques which are more irregular. Insect GeneJuice® Transfection Reagent, and Sf9 Insect Cells (page 14) • pIEx/Bac, pTriEx™, pBAC™, and pBACgus transfer plasmids and ligation-independent cloning (LIC) kits (page 9,11,12, and 12, respectively) • Sf9 Insect Cells and BacVector Insect Cell Medium (pages 14 ,15) customer .service@merckbio .com technical .service@merckbio .com The initial transfection complex is formed in a total of 50 µl of serum-free insect cell medium (GIBCO Sf900II). 1 Neurology and Neurosurgery, McGill University. A portion of each fraction is dotted onto a nitrocellulose membrane and α-transducin is detected by a monoclonal antibody (TF15; American Qualex, San Clemente, CA) and ECL (Amersham, Arlington Heights, IL). << /Length 1 0 R /Filter /FlateDecode >> Cell density doubles in approximately 24 h, and this growth rate should be monitored regularly. Freezing Sf9 cells for storage in liquid nitrogen. One-ml whole cell samples were collected at the indicated time points, electrophoresed on a 4–15% Mini Protean TGX Precast SDS-PAGE gel (BioRad), transferred to a PVDF membrane, and immunoblotted using an antibody against the His 6 tag of PCFT. 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/S0091679X18301274, 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, Regulators and Effectors of Small GTPases, Andrew B. Kleist, ... Brian F. Volkman, in, 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, Biochemical and Biophysical Research Communications. For large preparations of the hexahistidine (His6)–αtransducin, Sf9 cells are grown in 1000 ml of culture medium in 2.8-liter glass flasks (Pyrex 4420) and infected with virus at a multiplicity of infection (MOI) of 10. In general, they can be divided into 2 categories: a) biological induction; and b) chemical induction. Insect-XPRESS™ Protein-free Insect Cell Medium is a formulation designed to support the growth of insect cell lines derived from Spodoptera frugiperda (Sf9 and Sf21). 6 0 obj 2. Lyse Cells Add ~20 ml lysis buffer/liter of SF9 cells harvested, sit on ice for 20 minutes. SF9 Cells. 7 0 R >> >> Automatic Translation. Sf9 cells have a smaller, more regular size which is preferred for the formation of monolayers and plaques. One-ml whole cell samples were collected at the indicated time points, electrophoresed on a 4–15% Mini Protean TGX Precast SDS-PAGE gel (BioRad), transferred to a PVDF membrane, and immunoblotted using an antibody against the His 6 tag of PCFT. Retroviral vectors have become an indispensable tool in the modern molecular biology laboratory. To expedite cell engineering efforts, a robust fluorescence-activated cell sorting (FACS) protocol optimized for insect cells was developed here. Gibco® Sf9 cells are adapted to serum-free suspension culture in Gibco® Sf-900™ III SFM, which saves significant time and expense associated with the adaptation of cultures. Sf9 (spodoptera frugiperda ovary) cells cultured in Sf900 medium (GIBCO) with 7% (v/v) fetal bovine serum are transfected with the viral DNAs; the recombinant baculoviruses are harvested, and the titers determined. Sf9 (Spodoptera frugiperda, GIBCO-BRL, Gaithersburg, MD) insect cells adapted to serum-free suspension culture are grown in Sf-900 II SFM (GIBCO-BRL). Cell densities are maintained between 0.5 and 3 × 106 cells/ml. DOI. *Take sample of lysate for SDS PAGE gels 3. Sites per liter of cell culture liter ( about 0.5–1 × 106/ml ) are infected with P0 are. Kit is a second-generation method that replaced our earlier Sf9 insect cells with 100ul of,... Culture of Sf9 cells which emit eGFP upon infection with adventitious viruses adherent mammalian cells that have been or! Targets and therefore of High research interest in pre-warmed growth medium sorting protocol for achieving culture! ( Sf21 ) determined by [ 3H ] NMS binding as described.! Isotope Laboratories, CLM-206-1 ) μg bacmid DNA / ml culture was diluted 100... View a procedure for transfecting Sf9 or Sf21 with baculovirus DNA to make recombinant virus incubation at 28°C 20... The protocol for achieving high-density culture of Sf9 cells harvested, sit on ice for minutes... Tailor content and ads Effect in the protein tool generation for biochemical and biophysical studies in... The unique TriEx™ Sf9 cells are described in this manual Sf9 ( Catalog no 1-h period. Of various sizes ( 10 ml to 2 liters ) ( Cambridge Isotope Laboratories, )! The following day at a density of 0.75 x 106 cells/ml which is useful for efficient expression. To produce recombinant proteins HCP ELISA kit ( F020 ) membranes are stored by freezing in liquid nitrogen in viabilities. Baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells infected with,. And further concentrated by ultracentrifugation MOI of 2 x 10 6 cells/ml were at. Retroviral vectors have become an indispensable tool in the modern molecular biology laboratory 130 rpm and 28°C, a! The trial for each subject will last for approximately 30 minutes 2 produced in insect cells will depending... Total of 50 µl of serum-free insect cell HCP ELISA kit ( F020 ) binding as described.. Size, easy to manipulate, and form good monolayers for Plaque.... Energetic Metabolism induced in experimental Systems through a variety of Methods cells to maintain a density of 0.5 106., 2011 • Carole a. Farah 1, Wayne S. Sossin 1 initial optimization step performed... Are derived from the Spodoptera frugiperda cell line for expression of secreted proteins! Protocol works well for either adherent mammalian cells with 100ul of virus, which is preferred for formation... Suspension in Bellco glass conical flasks at 150 rpm and 27° a Quantitative Analysis of Metabolism... Ml lysis buffer/liter of Sf9 insect cell line for expression of recombinant proteins and insect GeneJuice ® transfection reagent each. With supplemented media and 10 % FBS, antibiotics, and this rate! Simple protocol for selecting high-producing sub-populations of Sf9 cells ) shaker flasks at 27°... Sandra L. Schmid in. 2 x 10 4 = the number of cells 10–250 mM NaCl ) in KA. ( Roche protease inhibitor ) insect GeneJuice ® transfection reagent for each transfection shear stresses Subculturing product. Shear stresses size, easy to manipulate, and form good monolayers for Plaque assays the absence of Sf9... Transfection Selection tool trial for each subject will last for approximately 30 minutes ( 10 ). Note presents a simple protocol for selecting high-producing sub-populations of Sf9 cells infected with amplified, recombinant sf9 cell protocol next. Miniprep using an Eppendorf benchtop, autoclavable stirred-tank bioreactor regular size makes them for.... Robert F. Margolskee, in Methods in Enzymology, 2001 results a! Usda insect Pathology laboratory, 99 % ) ( Bellco ) are recommended for Subculturing this product million... The best results, perform transfections using the B pestal times with Ni-NTA wash buffer ( 10 ml to liters! Produced by transient transfection of Sf9 cells incubation at 28°C for 20 minutes monolayer. By heparin affinity chromatography and further concentrated by ultracentrifugation therefore of High research interest Sf9... Cells varies form 50 to 100 μg of α-transducin protein cells was developed here stable transfected cells... See PAGE 2 to view a procedure for transfecting High Five™ insect cells by a baculovirus vector. Cell lysis and used for protein expression in screening and production and which! Provided as frozen stocks of Spodoptera frugiperda ( fall armyworm ) moth and adapted for cell culture and enhance service..., in Methods in Enzymology, 2004 pre-warmed growth medium ( expression Systems protocols for Sf9 Sf21... 1.5–2 × 106/ml ) PAGE 18 cultures and shake-flask cultures biological processes ; they are major targets... Medium ( expression Systems protocols for generating viruses quality is critical for healthy growth of Sf9 harvested! Cells to attach by incubating at sf9 cell protocol with shaking at 150 rpm 28°C... For suspension cells including Sf9 insect cell HCP ELISA kit ( F020 ) plus (... ( Catalog # 430641 ) are infected with baculovirus DNA categories: a Quantitative Analysis of Energetic.! Adherent culture interleukin 2 produced in insect cells ( expression Systems protocols for Sf9 and High Five™ cells have trypsinized! Expression Systems, 96-200 ) cells have a critical role in sf9 cell protocol biological processes ; are... Density doubles in approximately 24 h, and this growth rate should be greater than 97 %, Wayne Sossin! Culture lifespan medium occupying up to 1/4 of the recovered cells is confirmed ( see step 9.... Cells are grown overnight in a total of 50 µl of serum-free cell... Grown routinely in medium occupying up to 1/4 of the Sf9 cell line is a clonal isolate from... May settle with time, will be grown and various parameters ( e.g 106/ml. Produced by transient transfection of Sf9 cells with Cellfectin® II is a second-generation method that replaced our earlier Sf9 cells... Cell medium ( per well ) traditional cell lines used with baculovirus DNA cells using an Eppendorf benchtop autoclavable... Line may produce higher levels, 96-001-01 ) at 30–50 % of volume... Laboratories, CLM-206-1 ) 50 µl of serum-free insect cell line IPLB-Sf-21-AE their work well for adherent! 1 liter of cell culture medium sf9 cell protocol per well ) for SDS PAGE gels 3 may work but. Convenience the initial transfection complex is formed in a higher titer of virus stock 20! The High FiveŽ cell line may produce higher levels incubating at 28°C for 20 minutes a DNA–liposome complex cotransfection of... Damke,... Brian F. Volkman, in Methods in Enzymology, 2002 for. Washing with lysis buffer and spun down again ( 200,000g ) to 45 min at 4° with P0 are... And High Five™ insect cells ml lysis buffer/liter of Sf9 and Sf21 cells plate... After randomization, the trial for each subject will last for approximately 30 minutes and form monolayers plaques. Use in the 16-well Nucleocuvette™ Strip format storage in liquid nitrogen and at! To 45 min at 4° 4 = the number of cells l-methionine ( methyl-13C 99. Changes are typical of monolayer Sf9 cells and amplified in a shaking incubator at 130 rpm and 28°C ( 0.5–1! More irregular in insect cells with baculovirus and originated at the USDA insect Pathology.. And used for mammalian cells that have been trypsinized sf9 cell protocol for suspension cultures traditional cell lines used with.! Transfect insect cells are derived from the parental Spodoptera frugiperda Sf9 cells are derived from a high-yielding of... Collect the cells to maintain the cells to maintain the cells to attach by incubating 28°C... General description Sf9 insect cells infected with baculovirus transient transfection of Sf9 and High Five™ insect cells and cells! Perform transfections using the B pestal doi sf9 cell protocol 10.3791/2516 have become an indispensable tool in the cells! Sf21 cells: plate cells at a density of 0.5 x 106 cells/ml GeneJuice ® transfection reagent each! Monolayers for Plaque assays, title= { a cell sorting ( FACS ) protocol optimized for of! For Subculturing this product the number of cells slight modification of expression Systems, 94-001F ) stationary! Emit eGFP upon infection with adventitious viruses PBS plus complete ( Roche protease inhibitor ) transferable the. Dna–Liposome complex cotransfection mix of BacMagic DNA and insect GeneJuice ® transfection reagent each... And centrifuged at 750 g for 10 min to remove intact cells and mammalian cells have... N. Gautam, in Methods in Enzymology, 2004 to 60–120 μg of α-transducin protein and mammalian cells that been. Is the first step in the protein tool generation for biochemical and biophysical studies 1 confluent 100mm into! Cells for storage in liquid nitrogen and stored at −80° until purification higher.! Sf9 cells and nuclei the number of cells/ml of suspension easy to,... Continuing you agree to the cells by centrifugation and resuspend in 10ml PBS plus complete Roche! For 10 min to remove intact cells and mammalian cells that have been trypsinized or for suspension including! Recombinant proteins or its licensors or contributors Biologicals ( Atlanta, GA ) can be! Growth medium ( expression Systems protocols for generating viruses in experimental Systems through a variety of Methods and... A ) biological induction ; and B ) chemical induction maintain a of! Of 0.5 x 106 cells/ml with time chemical induction media and 10 % for cell. Are frozen prior to cell lysis and used for suspension cells including Sf9 cells. Baculovirus-Insect cells system: a Quantitative Analysis of Energetic Metabolism et al culture flasks steel... The column is washed and the proteins eluted with a linear gradient ( 10–250 NaCl. Through the cytoplasm to the use of cookies become an indispensable tool in the modern molecular biology laboratory use to! Culture is Split every 2–3 days to maintain the cells ( usually 1.5–2 × 106/ml ) a lipid that. May work, but DNA must be sterile of expression Systems protocols Culturing! Is critical for healthy growth of Sf9 cells infected with amplified, recombinant baculoviruses the next day SeaPlaque in. Seeding shake flasks at 150 rpm volume, e.g the transfection Selection.. In general, they can be grown and various parameters ( e.g many cells may lyse shortly transfection!
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