ACKR3 was expressed in Sf9 cells as previously described (27 (link)). Briefly, cells were cultured in ESF921 media (Expression Systems) at 27°C with shaking at 140 rpm. The Bac-to-Bac Baculovirus Expression System (Invitrogen) was used to produce baculovirus containing the different ACKR3 and CXCL12 variants as previously described (27 (link)). Briefly, recombinant bacmids were incubated with 3 μL of Xtreme Gene Transfection Reagent (Roche) and 100 μL of transfection medium (Expression Systems) for 30 minutes. The mixture was added to 2.5 mL of Sf9 cells at a density of 1.3 × 106 cells mL−1 and the cells were incubated for 96 h with 300 rpm shaking at 27°C. Cells were pelleted using centrifugation and 400 μL of the resulting supernatant (P0 stock) was used to transfect 40 mL of Sf9 cells at a density of 2.5 × 106 cells mL−1. After 48 h the cells were centrifuged and the resulting supernatant was stored at 4°C until further use (P1 stock). Virus titers were determined using flow cytometry staining with a phycoerythrin (PE)conjugated GP64 antibody. To initiate protein expression P1 virus corresponding to ACKR3 alone or ACKR3 and CXCL12 (co-expression) was added to Sf9 cells at a density of ~2.5 × 106 cells mL−1 at a multiplicity of infection of 5. Cells were grown for 48h at 27°C with shaking at 140 rpm prior to experiments.
Transfection medium
Manufactured by Expression Systems
Sourced in Switzerland
Transfection Medium is a specialized cell culture medium designed to facilitate the introduction of foreign genetic material, such as DNA or RNA, into cells. It is optimized for efficient and controlled transfection, enabling researchers to study gene expression, protein production, and other cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Bac to bac baculovirus expression system, by Thermo Fisher Scientific (4 mentions)
X tremegene transfection reagent, by Roche (2 mentions)
Gp64 pe antibody, by Expression Systems (2 mentions)
X tremegene hp dna transfection reagent, by Roche (2 mentions)
Esf 921 media, by Expression Systems (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Bac to bac system, by Thermo Fisher Scientific (1 mentions)
5 protocols using transfection medium
1
ACKR3 Expression in Sf9 Cells
2
High-Titer Baculovirus Production for 5-HT2C Receptor Expression
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High-titer recombinant baculovirus (>109 viral particles per ml) was obtained using the Bac-to-Bac Baculovirus Expression System (Invitrogen). Recombinant baculovirus was generated by transfecting 5–10 μg of recombinant bacmid into 2.5 ml Spodoptera frugiperda (Sf9) cells at a density of 106 cells per ml using 5 μl of FuGENE HD Transfection Reagent (Promega) and Transfection Medium (Expression Systems). After 4 days of shaking at 27 °C, P0 viral stock with ~109 virus particles per ml was harvested and used to generate high-titer baculovirus stock. Viral titers were determined by flow-cytometric analysis of cells stained with gp64-PE antibody (Expression Systems) (Hanson et al.2007 (link)). Expression of the 5-HT2C receptor was carried out by infection of Sf9 cells at a cell density of 2–3 × 106 cells/ml with P1 virus stock at multiplicity of infection (MOI) of five. Cells were harvested by centrifugation at 48 h post-infection and stored at −80 °C until further use.
3
Baculovirus Expression of 5-HT2C Receptor
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The Bac-to-Bac Baculovirus Expression System (Invitrogen) was used to generate high-titer recombinant baculovirus (>109 viral particles per ml). Recombinant baculovirus was produced by transfecting recombinant bacmids (2.5–5 μg) into Spodoptera frugiperda (Sf9) cells (2.5 ml, density of 106 cells per ml) using 5 μl of X-tremeGENE HP DNA Transfection Reagent (Roche) and Transfection Medium (Expression Systems). After 4 d of shaking at 27°C, P0 viral stock (~109 virus particles per ml) was harvested as the supernatant of the cell suspension to produce high-titer viral stock. Viral titers were analyzed by flow cytometry on cells stained with gp64-PE antibody (Expression Systems). 5-HT2CR was expressed by infecting Sf9 cells at a cell density of 2–3 × 106 cells per ml with P1 virus at MOI (multiplicity of infection) of 5. Cells were harvested by centrifugation 48 hr post infection and stored at −80°C for future use.
4
Recombinant Protein Expression in Bacteria and Insect Cells
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A pET-28a vector containing the MSP1D1 gene construct was transformed into BL21-Gold (DE3) competent cells for growth and expression. Cultures were grown at 37 °C.
For GLP-1R and GCGR, recombinant baculoviruses were generated by transfecting recombinant bacmids into Spodoptera frugiperda (Sf9) cells, using X-tremeGENE HP DNA Transfection Reagent (Roche, Basel, Switzerland) and Transfection Medium (Expression Systems, Davis, CA, USA). Recombinant baculoviruses were amplified using the Bac-to-Bac system (Invitrogen, Carlsbad, CA, USA). Cultures were grown at 27 °C.
For GLP-1R and GCGR, recombinant baculoviruses were generated by transfecting recombinant bacmids into Spodoptera frugiperda (Sf9) cells, using X-tremeGENE HP DNA Transfection Reagent (Roche, Basel, Switzerland) and Transfection Medium (Expression Systems, Davis, CA, USA). Recombinant baculoviruses were amplified using the Bac-to-Bac system (Invitrogen, Carlsbad, CA, USA). Cultures were grown at 27 °C.
5
High-Titer Recombinant Baculovirus Production
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