Cell Cycle Analysis Kit

50T, 100T, 200T

Flow cytometry

Propidium iodide DNA staining for cell-cycle and apoptosis analysis

Flow cytometry excitation: 488 nm.

Staining Buffer and Propidium Iodide Solution (25×): 4 °C, dark.

Propidium iodide (PI) is a fluorescent dye for double-stranded DNA. Fluorescence is emitted when PI binds double-stranded DNA, and the fluorescence intensity is proportional to the amount of double-stranded DNA. After the intracellular DNA has been stained with PI, the cellular DNA content can be measured by flow cytometry, and cell cycle and apoptosis analyses can then be performed on the basis of the distribution of DNA content. With PI staining, if the fluorescence intensity of G0/G1-phase cells is taken as 1, then the theoretical fluorescence intensity of G2/M-phase cells, which contain a doubled set of genomic DNA, is 2, and that of S-phase cells, which are replicating DNA, falls between 1 and 2. In apoptotic cells, nuclear condensation and DNA fragmentation cause part of the genomic DNA to be lost during the staining procedure; apoptotic cells therefore show distinctly weak PI staining, i.e., a fluorescence intensity below 1, giving rise to the so-called sub-G1 peak (the apoptotic cell peak) in the flow cytometric fluorescence histogram. When cells undergo apoptosis, cytoplasmic and chromatin condensation, nuclear fragmentation, and the formation of apoptotic bodies change the light-scattering properties of the cells. In early apoptosis, the forward-angle light-scattering capacity of the cells decreases markedly, while side scattering increases or remains unchanged; in late apoptosis, both the forward- and side-scattering signals decrease. Apoptosis can therefore be monitored by measuring these changes in light scattering with a flow cytometer.

This kit is generally used for cell cycle and apoptosis analysis of cultured adherent or suspension cells. For cell cycle and apoptosis analysis of tissue samples, the tissue must first be digested into a single-cell suspension before the assay can be performed.

Each sample may contain 1 × 10⁵ to 1 × 10⁶ cells.

1. Preparation of cell samples

a) Suspension cells:

i. Collect the cells into a centrifuge tube and centrifuge at 1000–2000 rpm for 5 min; carefully remove the supernatant.

ii. Gently resuspend the cells in 1 ml of PBS pre-chilled to 4 °C, count the cells, centrifuge at 1000–2000 rpm for 5 min, and carefully aspirate the supernatant.

iii. Add another 1 ml of PBS pre-chilled to 4 °C to resuspend the cells, centrifuge at 1000–2000 rpm for 5 min, and carefully aspirate the supernatant.

b) Adherent cells:

i. Transfer the culture medium to a centrifuge tube, wash the adherent cells once with PBS, and add an appropriate volume of EDTA-free trypsin cell digestion solution to detach the cells.

ii. Incubate at room temperature until the cells can be detached by gentle pipetting, then aspirate the trypsin digestion solution. Avoid over-digestion with trypsin.

iii. Gently resuspend the cells in 1 ml of PBS pre-chilled to 4 °C, count the cells, centrifuge at 1000–2000 rpm for 5 min, and carefully aspirate the supernatant.

iv. Add another 1 ml of PBS pre-chilled to 4 °C to resuspend the cells, centrifuge at 1000–2000 rpm for 5 min, and carefully aspirate the supernatant.

2. Cell fixation

i. Add 1 ml of the cell suspension dropwise into 4 ml of 95% ethanol pre-chilled on ice with gentle vortexing (perform on ice), mix, and fix at 4 °C for 2 hours or longer. Fixation for 12–24 hours may give better results.

ii. Centrifuge at about 1000 rpm for 3–5 min to pellet the cells. For particular cell types, if the pellet is insufficient, the centrifugation time or force may be appropriately increased. Carefully aspirate the supernatant; about 50 µl of ethanol may be left behind to avoid aspirating the cells.

iii. Add about 5 ml of PBS pre-chilled on ice, resuspend the cells, and centrifuge again to pellet them; carefully aspirate the supernatant, leaving about 50 µl of PBS to avoid aspirating the cells. Gently flick the bottom of the tube to disperse the cells and prevent clumping.

3. Preparation of the PI staining solution: with reference to the table below, prepare an appropriate volume of PI staining solution according to the number of samples to be assayed:

Note: the freshly prepared PI staining solution may be stored at 4 °C for a short period and should preferably be used on the same day.

4. Staining: add 0.4 ml of PI staining solution to each tube of cells, resuspend the pellet slowly and thoroughly, and incubate at 37 °C protected from light for 30 minutes. Afterwards, the samples may be stored at 4 °C or on ice protected from light. Flow cytometric analysis should be completed within 24 hours after staining, preferably on the same day.

5. Flow cytometric detection and analysis: detect the red fluorescence with the flow cytometer at an excitation wavelength of 488 nm, and record light scattering at the same time. Analyze the cellular DNA content and light scattering with appropriate analysis software.

1. This kit is for research use only.

2. This kit requires a flow cytometer for detection; PBS and 95% ethanol must be provided by the user.

3. For small-volume reagents, centrifuge for a few seconds to collect the reagent at the bottom of the tube before opening the cap.

4. Propidium iodide (PI) is toxic. Wear gloves when handling it and avoid contact with skin, eyes, and mucous membranes.

5. All fluorescent dyes are subject to photobleaching; protect them from light as much as possible during storage and use to slow down fluorescence quenching.

For research use only.