2.5.2 The enzyme modified comet assay to detect DNA oxidation
An enzyme-modified comet assay was performed as described by
Collins et al. (2023), only with
peripheral blood of the mothers. For the measurement of oxidized
purines, there is an additional step included after the lysis: the
slides were washed three times with ice-cold buffer (0.1 M KCl, 0.5 mm
Na2 EDTA, 40 mm HEPES, and 0.2 mg/ml bovine serum albumin; pH 8.0) and
incubated for 40 min at 37°C with formamidopyrimidine DNA glycosylase
(FPG) (Uniscience®, 1:1000) or buffer. Further steps were performed as
mentioned for the comet assay under alkaline conditions (Collins et al.,
2023). Net FPG sensitive sites were calculate as the difference between
the samples incubated with FPG and the samples incubated with buffer;
Damage Index of samplex/FPG – Damage Index of
samplex/buffer.