Flow cytometry DNA content assays provide a rapid and credible method of analyzing DNA ploidy and cell proliferation scores in tumor specimens. Both preserved and fresh specimens are amenable to this technique, and the information obtained can be used in conjunction with traditional prognostic factors to biologically evaluate tumors in their early stages, including breast, colon, lung, and bladder cancers.
Principle
The basic principle of DNA content analysis of solid tumors is based on the use of flow cytometry to detect the information of DNA ploidy status and the cell proliferation fraction that can objectively reflect subcellular microscopic characteristics, and these indicators can be used for biological evaluation of human tumors.
Operation method
Analysis of DNA content in solid tumors
Principle
The basic principle of DNA content analysis of solid tumors is based on the use of flow cytometry to detect the information of DNA ploidy status and the cell proliferation fraction that can objectively reflect subcellular microscopic characteristics, and these indicators can be used for biological evaluation of human tumors.
Materials and Instruments
Equipment: Move The basic procedure for solid tumor DNA content analysis can be divided into the following steps: 1.1 Place the tissue in a 60 mm × 15 mm Petri dish. Add 2 ml of fresh cell culture medium or PBS. 1.2 Cut the tissue into suitable pieces using a #21 scalpel blade. Sieve the tissue through a 500 μm stainless steel mesh. 1.3 Transfer the cell suspension to a new tube after filtering through 35 μm nylon mesh (Small Parts, Miami Lakes, FL). 1.4 3 ml of PBS was added and mixed with a micro-sampler. 1.5 The cells were centrifuged in a benchtop centrifuge at 500 g for 5 min to remove the supernatant. 1.6 Resuspend cells with 3 ml PBS and mix well, repeat step 5. 1.7 Resuspend cells with 1 ml PBS and filter through a 35 μm nylon mesh. 1.8 For cell counting, use 3 × 105 cells to prepare a cell centrifuge slice using a cell centrifuge slice machine. 1.9 The remaining cell suspension was centrifuged at 500 g for 5 min to remove the supernatant. 1.10 Gently resuspend the cells in 1 ml of 0.9% saline. 1.11 Add 2.5 ml of cold 95% ethanol to the suspension at low speed. Place the samples at 4°C for at least 1 h until ready for analysis by flow cytometry. 1.12 For each sample, prepare two tubes as described below: Tube 1 = patient cells Tube 2 = 1:1 mixture of patient and normal lymphocytes. 1.13 Dispense a total of 1 × 106 cells fixed in 70% ethanol into appropriate tubes. 1.14 Add 3 ml PBS, centrifuge at 500 g for 5 min, remove supernatant and repeat. 1.15 Resuspend the cells in 800 μl of PBS. 1.16 Add 70 μl of RNAase and 50 μl of PI and incubate at 37 °C for 20 min. 1.17 Filter the sample through a 35 μm nylon mesh and place on ice.
① Flow cytometer
② Centrifuge
Reagents:
① 0.5 mg/ml propidium iodide (PI);
② 1 mg/ml of RNase A (RNase);
① 0.5 mg/ml propidium iodide (PI)
④ 0.25% trypsin, 0.5% pepsin;
⑤ 0.05 mg/ml pepsin inhibitor A. ⑥ Histo-Solv X-Ray (HSR);
⑥ Histo-Solv X or xylene substitute (Curtin Matheson Scientific, Houston, TX).
2.1 Initial Preparation
2.1.1 For each sample, prepare one 16 mm × 100 mm borosilicate tube and two 12 × 75 mm plastic tubes. Labels were attached to the top of the tubes as markers.
2.1.2 For each paraffin block, cut one to three 50 μm slices, the number of slices depending on the content of the tissue. The block should be left at room temperature.
2.1.3 Place the sections into 16 mm × 100 mm test tubes, handling them carefully to prevent fragmentation.
2.1.4 The tubes can be sealed and stored at 22 °C until the specimens are processed.
2.2 Day 1
Fill the tubes with the following solution to cover the tissue sections and incubate directly. At the end of the incubation, aspirate as much of the solution as possible without interruption so that the tissue sections remain at the bottom of the tube.
2.2.1 Histo-Solv X or xylene substitute, treat 3 times for 15 min each.
2.2.2 Add anhydrous ethanol and process 2 times for 10 min each time.
2.2.3 95% ethanol, 2 treatments of 10 min each.
2.2.4 80% ethanol, 2 treatments, 10 min each.
2.2.5 70% ethanol, 2 treatments of 10 minutes each.
2.2.6 Add 50% ethanol and treat overnight.
2.3 Day 2
2.1 Preparation of pepsin. Incubate the pre-dissolved pepsin in a 37℃ water bath for 30 minutes before use.
2.2 Remove ethanol from the tubes.
2.3 Resuspend tissue sections in PBS and rehydrate for 10 min. centrifuge at 500 g for 5 min. repeat once.
2.4 Remove the PBS and add 2.5-3 ml of pre-warmed pepsin.
2.5 Incubate the tubes in a water bath at 37 ℃ for 20-45 min.
2.6 Blow the tissue sections and pepsin with a 3-cc syringe and an 18-gauge needle 1 to 2 times.
2.7 Add 175 μl of pepsin inhibitor A to the test tube and mix well. Place the tube on ice.
2.8 Centrifuge at 500 g for 5 min.
2.9 Resuspend with 2-3 ml of PBS and centrifuge. Remove supernatant and repeat the procedure.
2.10 Filter through a 35 μm nylon mesh and transfer to a clean 12 mm × 75 mm plastic test tube.
2.11 For cell counting, adjust the cell concentration to 1 × 106/ml.
2.12 Transfer 900 μl of cell suspension to a second 12 × 75 mm plastic test tube and add 100 μl of pre-warmed RNAase and incubate at 37 ℃ for 30 min. store the remaining cell suspension at -20 ℃.
2.13 Add 50 μl of PI to the tubes and place in the refrigerator for 30 min or on ice.
3 Analysis3.1 Instrument operation and data acquisition: The flow cytometer is adjusted to use blue light as excitation light and a red filter for detection. 488 nm laser excitation is used for PI, and the emission spectrum can be detected by a >610 nm long-wave filter. The instrument was operated in accordance with the manufacturer's operating procedures, emphasizing that the instrument should be calibrated daily with calibration pellets (beads) to obtain consistent resolution and peaks on the detection channel. The linearity of the photomultiplier tubes (PMTs) is checked once a month. The assay should be pre-constructed to capture the forward scattered light, DNA peaks, and fluorescence signals in their entirety and width to determine the G2/M phase of dividing cells.
3.2 Controls: For fresh solid tumor analysis, normal peripheral blood lympho-cytes (NPBLs) are used as a biological control to evaluate the quality of the PI staining and the consistency of the G0/G1 phase of the diploid cells on the assay channel. The assay is performed after mixing equal amounts of tumor cells and normal control cell suspensions; tumor cells can be evaluated by ploidy number.
3.3 Determination: DNA index and cell cycle data can be obtained by commercial software or manually. For manual analysis, the cursor area can be set at the G0/G1, peak and the S + G2/M area. the DNA Index is calculated by dividing the G0/G1, Heterozygous peak (x-axis) on the assay channel by the G0/G1, Diploid peak on the assay channel for the endogenous reference. By definition, the diploid DNA index (DI) should be equal to 1.00. S or S + G2/M cell cycle analysis is usually calculated using the amount of cells (area) at each cell cycle interval divided by the total number of cells.
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