(A) Saliva NLP, obtained as in the
Materials and Methods
, were inoculated into serum-free DMEM for 1 day (lane 1), 2 days (lane 2), and 14 days (lane 3). NLP were then pelleted, washed, and introduced into DMEM containing 5% HS, and incubated for 1 day (lane 4) and 12 days (lane 5). (B) Saliva NLP were inoculated into DMEM containing 5% HS for 4 h (lane 1), then pelleted, washed, and inoculated into serum-free DMEM for 1 day (lane 2), 2 days (lane 3), 5 days (lane 4), and 6 days (lane 5). (C) Urine NLP inoculated into serum-free DMEM and incubated for 1 day (lane 1), 2 days (lane 2) and 4 days (lane 3), after which NLP were collected, washed, and reinoculated into DMEM containing 5% HS and incubated for 2 days (lane 4). Formation of NLP from saliva and urine, their processing and subculturing, and standardization of the protein contents loaded onto each lane are described in the
Materials and Methods.
Note the gradual fading of protein bands in serum-free medium as well as the acquisition of multiple protein bands in the presence of serum.