This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.

Operation method

Optical and Fluorescence Microscopy

Materials and Instruments

Cell suspensions
Lenkostat Hoechst 33258 Acridine Orange Ethidium Bromide
Optical microscope Fluorescence microscope

Move

I. Lenkostat staining of spinning cell preparations

1. Add 100 μl of cell suspension to the rotary chamber and centrifuge at 500 r/min for 2 minutes. Air dry the slides for at least 5 minutes.

2. Fix cells for 10 seconds in Lenkostat fixative: 2 mg/ml malachite green in 100% methanol. Can be removed dropwise.

3. soak slides in Lenkostat solution 1 for 10 seconds to stain cytoplasm: 0.1% eosin; 0.1% formaldehyde; 0.4% disodium phosphate; 0.5% potassium dihydrogen phosphate. Can be blotted dry with paper towels.

4. Re-stain the cytoplasm by immersing the slide for 10 seconds in Lenkostat solution 2: 0.04% methylene blue; 0.04% azure A; 0.4% disodium phosphate; 0.5% potassium dihydrogen phosphate. Rinse off excess dye and air dry slides.

5. Observe under a light microscope to determine if the desired staining is obtained. If so, a slide specimen may be prepared with Pemount.

6. To determine the number of apoptotic cells, they should be viewed at 40x magnification. At least 3 fields of view including approximately 100 cells should be analyzed to give the percentage of apoptosis and necrosis.

Hoechst 33258 Staining of Suspended Cells

1. Incubate 100 μl of suspended cells with 1 μl of Hoechst 33258 for 10 min.

2. Add the sample to a rotary chamber, centrifuge at 500 r/min for 2 minutes, air-dry and cover-slip the cells to minimize light diffraction, and count at least 300 cells with a fluorescence microscope equipped with a DAPI filter, using a 60x objective.

Acridine Orange/Ethidium Bromide (AO/EB) Staining

1. Incubate 25 μl of suspended cells with 1 μl of AO/EB solution. Mix gently. Each sample should be mixed before micro quantification. Samples must be measured immediately.

2. Place 10 μl of suspended cells on a microscope slide, cover the slide, and examine at least 300 cells with a fluorescence microscope equipped with a fluorescence filter and a 60x objective.

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