This experiment is based on "Color Atlas of Practical Flow Cytometry", edited by Shukui Wang and Zhenying Zhou.

Operation method

Preparation and analysis of cellular RNA content samples

Materials and Instruments

Single Cell Suspensions
PY Reagent Acetic acid buffer Phosphate buffer DNA enzyme digest

Move

I. Reagent Preparation

1. Preparation of PY reagent: 1.0 g of PY should be dissolved in 100 ml of distilled water to form a master batch, and stored in the refrigerator at 4 ℃. PY working solution: Take 1 ml of PY master batch, add 100 ml of 1 mol/L acetate buffer of pH 4.7, and dilute it to a concentration of 0.01%.

2. 1.0 mol/L pH 4.7 acetic acid buffer: 11.55 ml of glacial acetic acid and distilled water to 1000 ml; 16.4 g of anhydrous sodium acetate and distilled water to 1000 ml; 255 ml and 245 mI of the above two liquids were taken respectively, and 200 ml of distilled water was added to make it.

3. Prepare phosphate buffer of pH 7.2.

4. Preparation of DNA enzyme digestion solution: DNA enzyme 0.05 mg/ml, 0.25 mol/L citric acid, 2.5 mol/L TrisHCl, pH 6.0.

II. Staining Procedures

1. Prepare single cell suspension at ^1x106/ml.

2. Add 0.5 ml of DNA enzyme solution, digest at 37℃ for 20 min, and discard the DNA enzyme.

3. Add 1.0 ml of 0.01% PY working solution, stain for 30 min at room temperature, and centrifuge to precipitate (800 r/min for 5 min).

4. Remove the staining solution, wash with PBS 3 times, then add 1.0 ml of PBS and test on the machine.

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