Seven-week-old female BALB/c mice were purchased from NARA Biotech (Seoul, South Korea) and maintained in approved facilities under specific-pathogen-free conditions. Spodoptera frugiperda (Sf9) cells were cultured in spinner flasks with SF900II media (Invitrogen, Carlsbad, CA, USA) at 27 °C, 140 rpm. T. gondii ME49 was maintained in BALB/c mice through serial passaging. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and IgA were purchased from Southern Biotech (Birmingham, AL, USA). Serially passaged Trichinella spiralis larvae were acquired from mice. Briefly, T. spiralis-infected murine muscle tissues were digested in pepsin-HCl solution, and larvae were manually counted under the microscope after repeated washing. Mice were infected with 150 T. spiralis larvae via oral gavage and immune sera were collected at 4 weeks post-infection, which were subsequently used as a negative control.
Sf900ii media
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SF900II media is a cell culture medium designed for the growth and maintenance of insect cells. It provides the necessary nutrients and supplements to support the proliferation of insect cell lines in suspension culture. The SF900II media is a versatile solution for researchers and scientists working with various insect cell-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Superdex 200 column, by GE Healthcare (5 mentions)
Pacgp67 a vector, by BD (5 mentions)
Nickel agarose, by Qiagen (4 mentions)
Biowhittaker insect xpress media, by Lonza (4 mentions)
Balb c mice, by Nara Biotech (2 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg and iga secondary antibodies, by Southern Biotech (1 mentions)
Fluorophore conjugated antibodies, by BD (1 mentions)
Pbacpak9 vector, by Takara Bio (1 mentions)
Fugene, by Promega (1 mentions)
Cellfectin 2 reagent, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Pib v5 his vector, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000 spectrometer, by Thermo Fisher Scientific (1 mentions)
Insect xpress media, by Lonza (1 mentions)
Nickel nta agarose, by Qiagen (1 mentions)
11 protocols using sf900ii media
1
Establishing Murine Infection Models
2
Recombinant human IL-2 variants production
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Example 3
Human IL-2 variants (amino acids 1-133), the IL-2Rβ ectodomain (amino acids 1-214), and γc (amino acids 34-232) were cloned into the pAcGP67-A vector (BD Biosciences) in frame with an N-terminal gp67 signal sequence and C-terminal hexahistidine tag and produced using the baculovirus expression system. Baculovirus stocks were prepared by transfection and amplification in Spodoptera frugiperda (Sf9) cells grown in SF900II media (Invitrogen), and protein expression was carried out in suspension Trichoplusia ni (High Five™) cells grown in BioWhittaker® Insect XPRESS™ media (Lonza). Proteins were expressed and captured from High Five™ supernatants after 48-60 hr by nickel agarose (QIAGEN), concentrated and purified by size exclusion chromatography on a Superdex™ 200 column (GE Healthcare), equilibrated in 10 mM HEPES (pH 7.2) and 150 mM NaCl. IL-2 variants used in SPR and cell based assays were expressed fully glycoslylated. For biotinylated receptor expression, IL-2Rβ and 7e were cloned into the pAcGP67-A vector with a C-terminal biotin acceptor peptide (BAP)-LNDIFEAQKIEWHE and hexahistidine tag. Receptor proteins were coexpressed with BirA ligase with excess biotin (100 uM).
3
Maintenance and Production of T. gondii
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Parasites and cells used in the present study were maintained as previously described (Kang et al., 2020b (link)). T. gondii ME49 strain was maintained by serial passage in BALB/c mice and ME49 cysts were isolated from the brains. Spodoptera frugiperda (Sf9) cells used for rBV production were cultured in spinner flasks with serum-free SF900II media (Invitrogen, Carlsbad, California, USA) at 27°C, 130–135 rpm. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and IgA secondary antibodies were purchased from Southern Biotech (Birmingham, AL, USA).
4
Plasmodium berghei Infection Model in BALB/c Mice
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BALB/c mice (six-week-old, female) were purchased from NARA Biotech (Seoul, Korea). Plasmodium berghei ANKA strain 2.34 was serially maintained in mice through intraperitoneal passaging. Recombinant baculovirus (rBV) and VLPs were generated using Spodoptera frugiperda Sf9 cells. The Sf9 cells were cultured in spinner flasks with SF900II media purchased from Invitrogen (Carlsbad, USA). P. berghei whole antigens were prepared following the method previously described [15 (link),16 (link),23 (link),24 (link)]. Blood samples were collected from P. brerghei-infected mice at 8 weeks post-infection via retro-orbital plexus puncture for serum acquisition. Monoclonal anti-M1 antibody and horseradish peroxidase (HRP)-conjugated secondary mouse IgG antibodies were purchased from Abcam (Cambridge, UK) and Southern Biotech (Birmingham, AL, USA), respectively. Fluorophore-conjugated antibodies were purchased from BD Bioscience (CA, USA) and used to perform flow cytometry.
5
Production and Purification of IL-2 Variants and Receptors
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Example 3
Human IL-2 variants (amino acids 1-133), the IL-2Rβ ectodomain (amino acids 1-214), and γc (amino acids 34-232) were cloned into the pAcGP67-A vector (BD Biosciences) in frame with an N-terminal gp67 signal sequence and C-terminal hexahistidine tag and produced using the baculovirus expression system. Baculovirus stocks were prepared by transfection and amplification in Spodoptera frugiperda (Sf9) cells grown in SF900II media (Invitrogen), and protein expression was carried out in suspension Trichoplusia ni (High Five™) cells grown in BioWhittaker® Insect XPRESS™ media (Lonza). Proteins were expressed and captured from High Five™ supernatants after 48-60 hr by nickel agarose (QIAGEN), concentrated and purified by size exclusion chromatography on a Superdex™ 200 column (GE Healthcare), equilibrated in 10 mM HEPES (pH 7.2) and 150 mM NaCl. IL-2 variants used in SPR and cell based assays were expressed fully glycoslylated. For biotinylated receptor expression, IL-2Rβ and γc were cloned into the pAcGP67-A vector with a C-terminal biotin acceptor peptide (BAP)-LNDIFEAQKIEWHE and hexahistidine tag. Receptor proteins were coexpressed with BirA ligase with excess biotin (100 uM).
6
Expression and Purification of EphA2 Kinase
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A construct of human EphA2 kinase domain (residues 595-897, Uniprot: P29317 ) was cloned into pBacPAK9 vector (Clontech) using EcoRI and XhoI sites using standard cloning techniques. Sf9 cells cultured in SF900II media (Invitrogen) supplemented with 100μg/ml penicillin and 100μg/ml streptomycin, were co-transfected with recombinant transfer vector containing EphA2 kinase domain and linearized viral DNA using Fugene (Promega). Supernatant containing the recombinant baculovirus were harvested 5 days post transfection. Viral stocks were amplified to obtain P2 stock. For protein expression, SF9 cells were cultured in suspension in SF900II media supplemented with 100μg/ml penicillin and 100μg/ml streptomycin to a density of 1.5×106 cells/ml. 400ml cells were infected with 10ml of P2 viral stock. Infected cells were harvested after 72 hours of incubation at 27°C and 120 RPM. Protein purification was carried out using standard Ni-affinity and size exclusion chromatography.
7
Expression and Purification of IL-2 Variants and Receptors
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Example 3
Human IL-2 variants (amino acids 1-133), the IL-2Rβ ectodomain (amino acids 1-214), and γc (amino acids 34-232) were cloned into the pAcGP67-A vector (BD Biosciences) in frame with an N-terminal gp67 signal sequence and C-terminal hexahistidine tag and produced using the baculovirus expression system. Baculovirus stocks were prepared by transfection and amplification in Spodoptera frupperda (Sf9) cells grown in SF900II media (Invitrogen), and protein expression was carried out in suspension Trichoplusia ni (High Five™) cells grown in BioWhittaker® Insect XPRESS™ media (Lonza). Proteins were expressed and captured from High Five™ supernatants after 48-60 hr by nickel agarose (QIAGEN), concentrated and purified by size exclusion chromatography on a Superdex™ 200 column (GE Healthcare), equilibrated in 10 mM HEPES (pH 7.2) and 150 mM NaCl. IL-2 variants used in SPR and cell based assays were expressed fully glycoslylated. For biotinylated receptor expression, IL-2Rβ and γc were cloned into the pAcGP67-A vector with a C-terminal biotin acceptor peptide (BAP)-LNDIFEAQKIEWHE and hexahistidine tag. Receptor proteins were coexpressed with BirA ligase with excess biotin (100 uM).
8
Recombinant Cytokine and Receptor Production
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Example 3
Human IL-2 variants (amino acids 1-133), the IL-2R13 ectodomain (amino acids 1-214), and γc (amino acids 34-232) were cloned into the pAcGP67-A vector (BD Biosciences) in frame with an N-terminal gp67 signal sequence and C-terminal hexahistidine tag and produced using the baculovirus expression system. Baculovirus stocks were prepared by transfection and amplification in Spodoptera frugiperda (Sf9) cells grown in SF900II media (Invitrogen), and protein expression was carried out in suspension Trichoplusia ni (High Five™) cells grown in BioWhittaker® Insect XPRESS™ media (Lonza). Proteins were expressed and captured from High Five™ supernatants after 48-60 hr by nickel agarose (QIAGEN), concentrated and purified by size exclusion chromatography on a Superdex™ 200 column (GE Healthcare), equilibrated in 10 mM HEPES (pH 7.2) and 150 mM NaCl. IL-2 variants used in SPR and cell based assays were expressed fully glycoslylated. For biotinylated receptor expression, IL-2Rβ and γc were cloned into the pAcGP67-A vector with a C-terminal biotin acceptor peptide (BAP)-LNDIFEAQKIEWHE and hexahistidine tag. Receptor proteins were coexpressed with BirA ligase with excess biotin (100 uM).
9
Heterologous Expression of Odorant Receptors
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Sf9 cells were cultured in an incubator (Thermo scientific, OH, USA) at a constant temperature of 24 to 25 °C. SF900-II media (Invitrogen, CA, USA) was used to culture cells. Sf9 cells were transfected with full coding sequences of cDNA of odorant receptors (AaOrco, AaOr8, and AaOr49) of Ae. aegypti, which were inserted into pIB/V5-His vector using Cellfectin® II Reagent (Invitrogen, Grand Island, NY, USA). Transfected cell lines were cultured in 10 μg/ml blastcidine SF900-II media (Invitrogen, Grand Island, NY, USA). Full length cDNAs were synthesized from mRNA extracted from the stylet with Superscript III (Invitrogen, CA, USA) using gene specific primers (Table S1). The expression vector plasmids were synthesized by inserting cDNAs of AaOrco, AaOr8, and AaOr49 into multiple cloning sites of a pIB/V5-His vector (Invitrogen, CA, USA) using the restriction enzymes NotI and XbaI (Koscamco, Anyang, Korea). Template pDNA for pIB-Or8 and Or49 and primer were then mixed with kit solutions and the total PCR reaction volume and conditions were followed as described earlier34 (link).
10
Culturing T. gondii and Sf9 Cells
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Parasites and cells were maintained and harvested as described previously [4 (link)]. Briefly, mice were intraperitoneally infected with T. gondii RH strain and ME49 strains. Tachyzoites of RH strain were separated from peritoneal fluids at 5 days PI and resuspended in phosphate-buffered saline (PBS), which were subsequently purified by discontinuous Percoll density gradient centrifugation. ME49 cysts were isolated from the brains of mice at 4 week PI. Spodoptera frugiperda (Sf9) cells were cultured and maintained at 27°C in spinner flasks at 130–135 rpm in serum-free SF900II media (Invitrogen, Carlsbad, California, USA) supplemented with 0.1% penicillin [16 (link)].
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