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Let $n$ count chromosomes in one complete set, and let $C$ denote the nuclear DNA amount in one unreplicated haploid set of this organism. Ploidy counts sets, whereas $C$ units count material; $C$ is not a universal mass shared by all species. Here we count a paired replicated chromosome as one until its sister chromatids separate. A copied diploid genome is therefore still diploid through S phase: $2n$ persists even as $2C$ becomes $4C$.

An original bookkeeping example uses a hypothetical diploid biological cell with $n=3$. Name its three homologous chromosome pairs $A,a$, $B,b$ and $D,d$. Assume ordinary nuclear DNA replication, no chromosome loss, and one continuous double-stranded DNA molecule per unreplicated chromosome or replicated chromatid. Count nuclear material only, excluding mitochondrion DNA.

For example, $A$ becomes the paired copies $A_1,A_2$ during S phase. They remain one replicated chromosome until separation. Each completed daughter receives one $A$ copy and one $a$ copy, and similarly for the other two pairs. Across both daughters, $2\times 6=12$ double-stranded DNA molecules and $2(2C)=4C$: division allocates the copied material without another round of DNA replication.

The whole undivided anaphase biological cell transiently contains $4n$ separately counted daughter chromosomes. This counting change does not establish a four-set ploidy in a completed daughter genome: each pole receives $2n$, and each completed daughter cell nucleus is diploid. Always state whether a number refers to one cell nucleus, one pole, or the entire undivided biological cell. Compare meiotic DNA content: after meiosis I, a haploid cell nucleus can contain $2C$, so amount alone cannot determine ploidy.

Under controlled staining and detector settings, integrated fluorescence can be approximately proportional to nuclear DNA amount. A histogram of accepted single-cell events then separates a $2C$ region, an intermediate region and a $4C$ region. These are ordinarily assigned to G0 phase/G1 phase, S phase, and G2 phase/M phase respectively in the stated diploid model.

A Cornell protocol fixes a single-cell suspension by adding cold 70% ethanol dropwise while mixing, reducing clumping. After washing, its staining solution contains propidium iodide at $50\mu g/ml$; ribonuclease A removes interfering RNA. It specifies excitation at $488\mathrm{nm}$ and emission around $600\mathrm{nm}$. The resulting readout is fluorescence per event, not a direct count of chromosomes. ^[ref]

Useful controls include an unstained sample for background and a reference population with known nuclear DNA amount, processed with identical settings. Two attached $2C$ biological cells can mimic one $4C$ event. Comparing signal integral with pulse height or width helps exclude such doublets; a gate selecting intact single particles also limits debris. These are proposed assay checks, not measurements underlying the illustration.

A $4C$ peak cannot distinguish G2 phase from mitosis; a $2C$ peak cannot distinguish G1 phase from G0 phase. Mixed ploidy, uneven staining and aggregates can invalidate phase assignments. Peak areas describe the accepted sample at collection, and do not by themselves establish phase durations, division rates or a particular arrest mechanism. A treatment comparison needs matched handling and another readout appropriate to the claimed mechanism.

Ploidy counts sets of chromosomes; DNA amount counts material. Let $n$ be the number of chromosomes in one complete set and $C$ the DNA amount of one unreplicated set. For the ordinary diploid model, read each row per completed cell nucleus, not as a total across all products:

In the illustrated $n=2$ example, the starting biological cell has four chromosomes. DNA replication gives eight chromatids, organized into four replicated chromosomes. Each product of meiosis I has two replicated chromosomes, hence four chromatids; each final product has two unreplicated chromosomes. Across completed products, $2(2C)=4C$ after meiosis I and $4(C)=4C$ after meiosis II: division redistributes the replicated DNA. A haploid cell nucleus can therefore have $2C$ without becoming diploid.