The coding sequence of the ATP sensor ATeam1.03YEMK (Imamura et al., 2009 (link)) was subcloned into the viral vector pSinRep5. Sindbis virus was produced as previously described (Piquet et al., 2018 (link)). Recombinant pSinRep5 and helper plasmid pDH26S (Invitrogen) were transcribed in vitro into capped RNA using the Megascript SP6 kit (Ambion). Baby hamster kidney-21 cells (BHK-21, clone 13, Mesocricetus auratus, hamster, Syrian golden), negative for mycoplasma contamination and purchased from ATCC (CCL-10, RRID:CVCL_1915 , lot number 1545545), were only used for viral production. BHK-21 cells were electroporated with sensor-containing RNA and helper RNA (2.107 cells, 950 µF, 230 V) and incubated for 24 h at 37 °C in 5 % CO2 in Dulbecco’s modified Eagle medium supplemented with 5 % fetal calf serum before collecting cell supernatant containing the viruses. The virus titer (108 infectious particles/ml) was determined after counting fluorescent baby hamster kidney cells infected using serial dilution of the stock virus.
Pdh26s
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The PDH26S is a laboratory centrifuge designed for general-purpose applications. It features a maximum speed of 4,000 rpm and a maximum relative centrifugal force of 2,800 x g. The centrifuge accommodates swing-out rotors and can handle sample volumes up to 400 mL.
Automatically generated - may contain errors
2 protocols using pdh26s
1
Producing Sindbis Virus Encoding ATP Sensor
2
Sindbis Virus Production Protocol
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The GA sequence (Genbank accession number: EF212028) has been described elsewhere (Drobac et al. 2010) and was similar to that of the G5A fusion protein reported previously (Baubet et al, 2000) . GA was inserted into the plasmid pSinRep5 (Invitrogen, Carlsbad, CA, USA) between SphI and ApaI sites. The resulting plasmid and the helper plasmid pDH26S (Invitrogen) were linearized with PacI and XhoI, respectively; and in vitro transcribed into capped RNA using the Megascript SP6 kit (Ambion, Huntingdon, UK).
Sindbis virus production was performed as described (Drobac et al. 2010) . BHK-21 cells (ATCC # CCL-10) were electroporated (20.10 6 cells/ml, 950 μF, 230 V) with recombinant and helper viral RNAs using the Gene Pulser II electroporation system (Bio-Rad Laboratories, Hercules, CA, USA) and grown 24 h at 37°C, 5% CO2 in DMEM containing 5% fetal calf serum before collecting the cell supernatant containing viral particles. The recombinant virus titer (1.8x10 8 ip/ml) was determined after counting fluorescent BHK cells infected using serial dilution of the virus stock.
Sindbis virus production was performed as described (Drobac et al. 2010) . BHK-21 cells (ATCC # CCL-10) were electroporated (20.10 6 cells/ml, 950 μF, 230 V) with recombinant and helper viral RNAs using the Gene Pulser II electroporation system (Bio-Rad Laboratories, Hercules, CA, USA) and grown 24 h at 37°C, 5% CO2 in DMEM containing 5% fetal calf serum before collecting the cell supernatant containing viral particles. The recombinant virus titer (1.8x10 8 ip/ml) was determined after counting fluorescent BHK cells infected using serial dilution of the virus stock.
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