Figure 9. Effect of serum, temperature, and EDTA on NLP formation.

NLP were prepared from 3 mM of CaCl₂, Na₂CO₃, and NaH₂PO₄ in DMEM containing different amounts of FBS or HS, as indicated, followed by incubation at 37°C for 1 day (A and B), 1 week (E and F), or 4°C for 2 weeks (C and D). Note that FBS had a stronger, dose-dependent inhibitory effect on NLP formation in all 3 situations when compared with HS. Inhibition could be seen for 2 weeks at 4°C, but at 37°C, it was overcome after 1 week, and this release of inhibition was more evident with HS. (G) NLP were prepared exactly as in Fig. 1B, by adding solutions of 0.25 M CaCl₂, Na₂CO₃, and NaH₂PO₄ (pH 7.4) to DMEM to the indicated concentrations in the presence of 10 mM EDTA, followed by incubation for 1 day. Addition of EDTA into the precipitation mix decreased the amount of NLP, except for well 6, containing 10 mM of precipitating reagents, indicating that excess EDTA is needed for the inhibition to work.