The existence of isotopes is direct evidence for the presence of neutrons in the nucleus, as $A$ can vary while $Z$ remains fixed.
How would you calculate the number of neutrons in the isotope $^{16}_8 \text{O}$?
When you assemble protons and neutrons to form a nucleus, the total mass of the nucleus is slightly less than the sum of the individual masses of its protons and neutrons.
This "missing mass" is called the mass defect, and it is converted into energy according to Einstein’s famous equation: $$
E = mc^2
$$
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