Peripheral blood from patients was collected in cell-free DNA collection tubes (Roche) at day one of the first administration of the systemic chemotherapy regimen as well as 4–6 weeks after the first blood sample. Blood samples were proceeded within 12 hours of collection via a 2-step centrifugation protocol. First, plasma was separated from the other blood components by centrifugation at 2000 x g for 20 minutes. After transferring the upper plasma layer to a new conical tube, it was respun at 3200 x g for 30 minutes to remove cell debris. Subsequently the resulting plasma supernatant was stored at −20 °C in 10 mL cryotubes (VWR) until DNA isolation. Circulating DNA isolation from 5–10 mL plasma was performed on the Chemagic 360 Instrument (Perkin Elmer) with the isolation kit CMG-1111 (chemagic cfDNA 10k Kit special H12) according to manufacturer’s instruction. Cell-free DNA was eluted in ~40 µL elution buffer. DNA quantification was performed with Qubit® dsDNA HS Assay Kit (Invitrogen) according to the instructions provided by the manufacturer and purity was determined by Agilent 2200 TapeStation System. Cell-free DNA was stored at −20 °C until further analysis.
Cryotubes
Manufactured by Avantor
Sourced in France
Cryotubes are laboratory containers designed for the storage and preservation of samples at ultra-low temperatures, typically in liquid nitrogen or freezers. They are made of materials suitable for cryogenic conditions and provide a secure and reliable means of storing samples, such as cell cultures, tissues, or biological specimens, for extended periods.
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Lab products found in correlation
2 protocols using cryotubes
1
Plasma-derived Cell-free DNA Collection
2
Y. lipolytica Strain JMY7019 Cryopreservation
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The Y. lipolytica strain JMY7019 used in this study was kindly provided by the Micalis Institute, INRAE (Jouy-en-Josas, France). Its construction has been described in an article on the extraction of violacein synthesized by this strain using anionic surfactants [22 (link)]. The working cell bank (microbial stock) was prepared as follows: a cell sample was aseptically taken from the agar plate using a sterile loop and placed in a sterile baffled flask filled with the optimized culture medium (see Section 3.2 for composition). The inoculated medium was then incubated for 24 h at 28 °C with shaking at 170 rpm stirring (Thermo Scientific MaxQ 6000, Aubervilliers, France). After 24 h of incubation, the cells were cryopreserved by adding 1 mL of the culture medium into the cryotubes (VWR, Rosny-sous-Bois, France), supplementing this volume with 1 mL of a 1:1 (v/v) glycerol/water mixture, and gently shaking the tube to homogenize the suspension. The tubes were stored at −80 °C in an ultra-low-temperature freezer (Panasonic MDF-U700VX, Couëron, France).
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