Celltracker CM-Dil (Synonyms: Live Cell Tracer CM-Dil)

This plan only provides a guide, please modify it to meet your specific needs.

1. Prepare Celltracker CM-Dil staining solution:

(1) Prepare dye storage solution: Dissolve Celltracker CM-Dil in DMSO and prepare a storage solution with a concentration of 1-5mg/mL. The stock solution was aliquoted and stored in the dark at -20 °C.

(2) Use an appropriate buffer (such as serum-free medium, HBSS or PBS) to dilute the storage solution, prepare a Celltracker CM-Dil working solution with a concentration of 1-5 μg/mL, and use a 0.22 μm filter to filter and sterilize.

Note: Please adjust the concentration of Celltracker CM-Dil working solution according to the actual situation. Prepare it now.

2. Cell suspension staining (taking 6-well plate as an example)

(1) Centrifuge suspended cells at 1000g for 3-5 minutes. Discard the supernatant and wash twice with PBS for 5 minutes each time.

(2) Wash the adherent cells twice with PBS, add trypsin to digest the cells, and centrifuge at 1000g for 3-5 minutes after digestion is completed.

(3) Add 1 mL of dye working solution to resuspend the cells, and incubate at room temperature in the dark for 15-30 minutes. The optimal culture time for different cells is different.

(4) After the incubation, centrifuge at 1000g for 5 minutes, remove the supernatant, and add PBS to wash 2-3 times, 5 minutes each time.

(5) Use serum-free cell culture medium or PBS to resuspend the cells and observe them through fluorescence microscopy or flow cytometry.

3. Cell adhesion staining

(1) Culture adherent cells on sterile coverslips.

(2) Remove the coverslip from the culture medium, aspirate the excess culture medium, add PBS to wash 1-2 times, and place the coverslip in a humid environment.

(3) Add 100 μL of dye working solution from one corner of the coverslip, shake gently to evenly cover all cells with the dye, and incubate at room temperature in the dark for 15-30 minutes.

(4) Aspirate away the dye working solution and wash the coverslip with PBS 2 to 3 times for 5 minutes each time.

4. Celltracker CM-Dil marks microbubbles (Microparticles or Microvesicles)

(1) Add an appropriate volume of Celltracker CM-Dil dye stock solution to the collected microbubble culture solution to reach a final concentration of 5 μM (or resuspend using 0.5-1ml Celltracker CM-Dil working solution), and use pipetting Gun and mix gently;

(2) Incubate at room temperature in the dark for 30 minutes, turning gently every 5 minutes.

(3) Transfer the incubated sample to a centrifuge tube, and use an ultracentrifuge to centrifuge at 16000-18000g and 4°C for 30 minutes;

(4) After centrifugation, carefully tilt and discard the supernatant in a clean bench. Use a micropipette with a sterile tip to remove the liquid that forms in the test tube but does not drip. This is the marked outer layer. secretion body;

Note: During this step, the free dye disappears with the decanted supernatant, so the solution can be decanted, but always be careful not to lose the precipitate.

5. Microscope detection: The maximum excitation/emission light of Celltracker CM-Dil is 553/570 nm.

Precautions:

1) Fluorescent dyes all have quenching problems. Please avoid light as much as possible to slow down fluorescence quenching.

2) For your safety and health, please wear a lab coat and disposable gloves.

3) This product can be used for fixed cells/tissues and tissue section staining, and can withstand fixation with 2% (w/v) paraformaldehyde and permeabilization with 0.2% (w/v) Triton X-100. Formaldehyde fixation, paraffin embedding, sectioning, xylene, graded ethanol and water cleaning have no adverse effects on staining.