n cell cycle analysis, particularly when using flow cytometry, an unstained control sample of MCF-7 cells (or any cell line) is typically used to establish a baseline for fluorescence and to help set gates for the analysis of stained samples.

Key Points Regarding Unstained Control in Cell Cycle Analysis:

1. Fluorescence Peaks: Unstained cells will not produce a peak in the fluorescence channel used for cell cycle analysis because they lack the fluorescent dye that binds to DNA (like propidium iodide or DAPI). However, they may still scatter light, producing a signal that can be detected as a baseline.

2. Background Signal: The unstained control may show some background fluorescence due to autofluorescence of the cells or other non-specific signals. This background should be minimal compared to stained samples.

3. Setting Gates: The purpose of the unstained control is primarily to help set the appropriate gates for identifying the different phases of the cell cycle (G0/G1, S, G2/M) in stained samples. It allows you to determine the threshold for positive staining.

4. Comparative Analysis: When analyzing stained samples, you will compare their fluorescence intensity against the unstained control to accurately quantify the proportion of cells in each phase of the cell cycle.

5. Cell Cycle Distribution: Stained MCF-7 cells will show distinct peaks corresponding to the different phases of the cell cycle (e.g., G0/G1, S, G2/M) based on their DNA content, while the unstained control should ideally not show these peaks.

Make sure you are using linear amplification for DNA histogram analysis. If possible also plot peak height vs peak area on your DNA signal so you can do doublet discrimination. Doublets will have the same peak area as G2/M cells but will have a lower peak height.

David G Spiller, I will consider your point when I repeat the experiment. Thank you so much.

Maryam Hataminejad .... Thanks a bunch for the thorough explanation! I really appreciate it!