Lipi-Deep Red

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

1. Preparation of Staining Solution

(1) Stock solution: Balance at room temperature for at least 10 minutes, briefly centrifuge, and dissolve Lipi-Deep Red in DMSO to prepare a 0.1 mM stock solution. After preparation, aliquot the stock solution and store it at -20℃ away from light for up to one month.

(2) Working solution: Dilute the stock solution with an appropriate buffer (such as serum-free medium, HBSS or PBS) to prepare a working solution of Lipi-Deep Red at a concentration of 0.1 - 0.5 μM.

Note:

① The working solution should be freshly prepared and used immediately.

② Serum-containing medium can also be used instead of serum-free medium.

③ For the best staining results, it is recommended to determine the optimal probe concentration and staining time based on the actual cell conditions during the initial experiment.

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 37℃ in the dark for 30-45minutes. 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, 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 37℃ in the dark for 30-45 minutes.

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

4. Observe using a fluorescence microscope or flow cytometer. Lipi-Deep Red has a maximum excitation/emission wavelength of 640/650-700 nm.

Note: If no fluorescence signal is observed, please try the following methods:

(1) Some cells have small lipid droplets. If it is difficult to confirm, it is recommended to increase the magnification of the fluorescence microscope.

(2) Extend the staining time to 1-2 hours.

(3) Increase the working solution concentration (increase the Lipi-Deep Red working solution concentration to 1 μM).

(4) You can add 200 μM oleic acid (cat. No. GC49917) to the cells and culture overnight as a positive control for observation and evaluation.

5. Staining steps for fixed cells:

It is recommended to fix cells with paraformaldehyde (PFA). Fixing cells with alcohols such as methanol is not recommended as it may affect the structure of lipid droplets. For some cell types, if there is no staining or insufficient staining intensity during the staining process, the optimal fixation conditions need to be explored.

(1) Example of staining after cell fixation (using HepG2 cells as an example)

a. Seed HepG2 cells in a Slide 8-well plate and incubate overnight in a 37℃ 5% CO₂ incubator.

b. Remove the culture medium and wash twice with PBS.

c. Add Lipi working solution prepared in PBS to the cells and incubate in a 37℃ incubator for 15 minutes.

d. Remove the supernatant and wash twice with PBS.

e. Add 4% PFA (in PBS) and fix at room temperature for 5 minutes.

f. Remove the supernatant, wash with PBS, and observe under a fluorescence microscope.

(2) Example of staining before cell fixation (using HeLa cells as an example)

a. Seed HeLa cells in a Slide 8-well plate and incubate overnight in a 37℃ 5% CO₂ incubator.

b. Remove the culture medium and wash twice with PBS.

c. Add 4% PFA (in PBS) and fix at room temperature for 5 minutes.

d. Remove the supernatant and wash twice with PBS.

e. Add Lipi working solution prepared in PBS to the cells and incubate in a 37℃ incubator for 30 minutes.

f. Remove the supernatant, wash with PBS, and observe under a fluorescence microscope.

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.