Cell Autophagy Staining Detection Kit (MDC)

I. Self-prepared materials:
Fluorescence microscope, low-speed centrifuge, EB staining solution, slides, coverslips
II. Operation steps (for reference only):
1. Staining of suspended cells:
(1) Dilute 10 μL of washing solution to 1 μL of washing solution with deionized water.
(2) Centrifuge at 800 g for 5 min, collect cells, wash cells once with 300-400 μL of 1 μL of washing solution, and discard the supernatant.
(3) Add an appropriate amount of 1 μL of washing solution to resuspend cells, count and adjust the cell concentration to 10⁶/mL.
(4) Take 90 μL of cell suspension to a new EP tube, add 10 μL of MDC staining solution, and mix gently. Stain at room temperature in the dark for 15-30 minutes. (See Note 1)
(5) Centrifuge at 800g for 5 minutes, collect cells, wash cells twice with 300-400 μL of 1× washing solution, and discard the supernatant.
(6) Add 100 μL of buffered collection solution to resuspend cells, drop onto a slide and cover with a glass slide.
(7) Observe under a fluorescence microscope (blocking filter wavelength 512nm), count and photograph.
2. Staining of adherent cells:
(1) Dilute 10× washing solution to 1× washing solution with deionized water, remove the culture medium and wash cells with 1× washing solution.
(2) Dilute an appropriate amount of MDC staining solution 10-fold with 1× washing solution, then add to the well plate and incubate at room temperature in the dark for 15-30 minutes. (See Note 2)
(3) After staining, wash twice with 1× cleaning solution, 3 minutes each time.
(4) Observe under an inverted fluorescence microscope (blocking filter wavelength 512nm), count and take pictures.
III. Staining results:
Normal cells:
Cells are uniformly stained with yellow-green fluorescence
Apoptotic cells:
Chromatin is condensed, the cell nucleus is fragmented into dots, and is stained with dense and intense green particles of varying sizes

Store at -20℃ away from light, valid for 12 months.