BODIPY 493/503 is a lipophilic fluorescent probe used to label cellular neutral lipid contents, displaying excitation/emission maxima of 493/503 nm, respectively.
BODIPY 493/503 (BODIPY) is an alternative lipid dye with lower background staining and narrower emission spectra[2]. BODIPY 493/503 was a convenient and simple approach to visualize lipid droplets (LDs) in both sectioned skeletal muscle and cultured adult single fibers. Furthermore, the dye was effective in both fixed and nonfixed cells, and the staining seemed unaffected by permeabilization[3]. The use of the fluorescent neutral lipid dye 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) to facilitate quantification of neutral lipid content by flow cytometry and observation of LDs (Lipid droplets) by microscopy[4]. BODIPY 493/503 contains a nonpolar structure that, upon binding to neutral lipid, emits a green fluorescence signal with a narrow wavelength range, making it an ideal fluorophore for multi-labeling experiments[5]. BODIPY 493/503 is well suited for quantitative analysis of LDs for high-throughput screening (HTS) applications[6]. BODIPY 493/503 produced an overall better staining for PHB (Poly [(R)-3-hydroxybutyric acid) than did Nile red[7]. Protocol from BODIPY 493/503 Staining of Neutral Lipid Droplets for Microscopy and Quantification by Flow Cytometryde novo lipogenesis[8] A. BODIPY staining for flow cytometry 1. Grow cells under culture conditions relevant for the study. A 35 mm dish/well is sufficient for the cell numbers required in this assay. For our assays, 50,000 A498 cells in 35 mm well were sufficient.
a. Overnight incubation of cells with 30 μM oleic acid can serve as a positive control for increased neutral lipid content, as oleic acid is a potent inducer of triglyceride synthesis and storage. Fatty acid free BSA serves as a control.
2. At the time-point of interest, prepare 2 μM BODIPY staining solution in PBS. The volume of staining solution required for each sample corresponds to the volume of media used for culturing cells. 3. Wash cells with a quick rinse using 3 ml PBS to remove media/serum. 4. Incubate on BODIPY staining solution in the dark for 15 min at 37 °C. Include an unstained control for flow cytometry. Note: From this point, protect samples from light as much as possible.
5. Wash cells with a quick rinse using 3 ml PBS to remove staining solution. 6. Trypsinize cells to generate a single cell suspension. For the A498 cell line used in this protocol, cells were incubated with Trypsin-EDTA (0.25%) for 5 min at 37 °C. 7. Add 5 ml of PBS and transfer cell suspension to a 15 ml conical tube. 8. Pellet cells at 250 × g, 5 min, 4 °C. 9. Aspirate supernatant, wash the cell pellet with a quick rinse using 3 ml PBS, and pellet cells at 250 × g, 5 min, 4 °C. 10. Carefully aspirate the supernatant and resuspend cells in 300 μl 1× flow cytometry buffer. 11. Pass cell suspension through a 35 μm filter into a FACS tube. 12. Perform flow cytometry. Obtain a minimum of 10,000 events per condition. 13. The investigator can analyze data as mean fluorescence or display the data as a histogram.
B. BODIPY staining for microscopy a) Autoclave coverslips in a glass bottle. b) In the tissue culture hood, place coverslips into 35 mm cell culture dishes. c) Prepare 2 mg/ml collagen solution in PBS. d) Treat the coverslips with collagen to promote cell adherence. Add 3 ml collagen solution to culture dishes and incubate at 37 °C for 30 min. Note: Use forceps to ensure that coverslips are flush with the bottom of the culture dish, eliminating any air bubbles that may be under the cover slips. e) Aspirate the collagen solution. f) Wash with PBS. g) Add PBS to culture dishes and place under UV light in the culture hood to sterilize. h) Plate cells into culture dishes containing the coverslips. The optimal cell number should be determined to achieve confluence of 30–50% at the time of staining to permit proper imaging. For A498 cells used in this protocol, 100,000 cells were plated in 35 mm wells to permit staining at 48 h post plating. i) Incubate under the culture conditions relevant to your experiment. i. For this protocol, A498 cells were incubated in DMEM (high glucose, L-glutamine, sodium pyruvate) supplemented with 10% FBS at 37 °C. ii. Overnight incubation of cells with 30 μM oleic acid with BSA can serve as a positive control for increased neutral lipid content, as oleic acid is a potent inducer of triglyceride synthesis and storage. Fatty acid free BSA serves as a control. j) At the time-point of interest, prepare 2 μM BODIPY staining solution in PBS. i. For this protocol, A498 cells were stained 48 h after plating, after an overnight incubation with BSA or BSA + oleic acid. k) Wash cells with 3 ml PBS. l) Incubate on 3 ml staining solution for 15 min at 37 °C. Note: From this point, protect samples from light as much as possible. m) Wash twice in 3 ml PBS. n) Fix cells in 3 ml 4% PFA for 30 min at room temperature. o) Remove 4% PFA. p) Wash samples 3 × 5 min in PBS. q) Use forceps to mount cover slips onto glass slides. i. Add a drop of Prolong® Gold antifade reagent with DAPI onto slide. ii. Use forceps to pick up cover slips and place onto the drop of mounting solution, ensuring that the side that side with cells is placed face down onto the mounting solution. r) Allow the mounting solution to cure overnight at room temperature. s) Slides can be stored at 4 °C or imaged immediately.
C. Recipes 1. 2 μM BODIPY staining solution a. Prepare 5 mM BODIPY stock solution: Dissolve 1.3 mg BODIPY in 1 ml DMSO and can be stored at −20 °C. b. 2 μM BODIPY staining solution can be prepared by diluting stock solution 1:2,500 in PBS.
2. 10× flow cytometry buffer 0.1 M HEPES (pH 7.4) 1.4 M NaCl 25 mM CaCl2 Notes: a. 10× buffer can be stored at 4 °C. b. Dilute to 1× using MilliQ water prior to use.
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