HCT116 WT and H1299 WT cells were purchased from the ATCC (Manassas, VA, USA). HCT116 cell lines over-expressing POR and the E. coli nitroreductases had been previously generated and validated for candidate gene expression 34 (link), 35 (link), 69 (link), in addition to the H1299 cell line over-expressing NfsA_Ec 54 (link) (Figure S1 ). Cell cultures were re-established from STR-authenticated frozen stocks every 3 months and were confirmed to be mycoplasma free by PCR enzyme-linked immunosorbent assay (ELISA) (Roche Diagnostics Corp, Basel, Switzerland). Cell lines were cultured in α-MEM using a humidified incubator (37 °C, 5% CO2) as previously described 70 (link), 71 (link) for a maximum of 12 weeks. Harvested cells were counted using an electronic particle counter (Z2 Coulter Particle Analyzer, Beckman Coulter, Florida, USA).
Z2 coulter particle analyzer
Manufactured by Beckman Coulter
Sourced in United States
The Z2 Coulter Particle Analyzer is a laboratory instrument used for accurately measuring the size and count of particles in a sample. It utilizes the Coulter principle to detect and analyze particles suspended in an electrolyte solution, providing precise data on particle concentration and size distribution.
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Lab products found in correlation
3 protocols using z2 coulter particle analyzer
1
Cell Line Generation and Maintenance
2
Cloning and Transfection of POR Gene
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Cells were maintained in culture under humidified atmospheric conditions with 21% O2 and 5% CO2 [75 (link),76 (link)] as described with < 3 month cumulative passage from authenticated stocks. MDA-MB-231 cells were sourced from Manchester University (UK) and STR phenotyped to confirm authenticity (CellBank, Sydney, Australia). Harvested cells were counted with an electronic particle counter (Z2 Coulter Particle Analyzer, Beckman Coulter, Miami, FL, USA). Sequences encoding POR, either the full length gene or with modified localisation sequences, were cloned into F527, a modified version of pEF-IRES [77 (link)]. Transfected cells were selected using puromycin and individual clonal cell lines generated.
3
Stable Cell Line Generation and Culture
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HCT116 WT cells were purchased from the ATCC (Manassas, VA, USA) and H1299 cells were sourced from Onyx Pharmaceuticals (California, USA). The open reading frame encoding NmeNTR was cloned into the Gateway compatible vector F279-V5 and cells were stably transfected using FuGene 6 (Roche, Basel, Switzerland) as previously described [24] . Cell cultures were re-established from STRauthenticated frozen stocks every 3 months and confirmed to be mycoplasma free by PCR enzyme-linked immunosorbent assay (ELISA) (Roche Diagnostics Corp, Basel, Switzerland). Neoplastic cell lines were cultured in α-minimal essential medium using a humidified incubator (37 °C, 5% CO 2 ) as previously described [13, 15] for a maximum of 12 weeks. Harvested cells were counted with an electronic particle counter (Z2 Coulter Particle Analyzer, Beckman Coulter, Florida, USA).
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