Oil red o staining kit

The lipid droplets in pre-adipocytes were stained using an Oil Red O staining kit (Solarbio, China) according to the manufacturer instructions. Briefly, cells were washed using PBS and fixed using 4% formaldehyde for 30 min. Then, the fixed cells were washed using 60% isopropanol for 30 sec and stained with Oil Red O solution (solutions A and B). After 20 min, the cells were rinsed using 60% isopropanol to remove the residual Oil Red O and repeatedly washed using distilled water. Stained cells were observed using a microscope.

The oil red O staining kit (G1262; Solarbio, Beijing, China) was used for oil red O staining. Briefly, the cells were washed with PBS buffer solution, fixed with cytochrome fixative for 30 min, washed with 60% isopropanol, stained with Oil Red O staining solution for 10–15 min, and then counterstained with Mayer haematoxylin solution for 1–2 min. Finally, the cell images were collected under an inverted microscope (IX-73; Olympus) to observe lipid droplets.
The triglyceride content of the cells was determined using the Prelude Triglyceride Determination Kit (Applygen Technologies, Beijing, China). The cells were washed with PBS three times and lysed in lysis buffer (R0010; Solarbio). TAG content was determined by enzymatic colourimetry at 570 nm using a microplate reader (iMark; BIO-RAD, Hercules, CA, USA).

The high-fat diet containing 60% calories from fat (D12492) was purchased from Xietong Pharmaceutical Bio-engineering Co., Ltd. (Jiangsu, China). Asparagus cochinchinensis roots were purchased from Kangdi Medicine Industry Co., Ltd. (Chongqing, China, Lot: 211101) and were authenticated corresponding authors. Glucose solution was purchased from Tisansheng Pharmaceutical Co., Ltd. (Hubei, China). Insulin was purchased from Novo Nordisk Pharmaceutical Co., Ltd. (Tianjin, China). Commercial kits for total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Hematoxylin and eosin (H&E) staining kit and Oil Red O staining kit were both purchased from Solarbio Science and Technology Co., Ltd. (Beijing, China). Cluster of differentiation 68 (CD68) and adhesion G protein-coupled receptor E1 (F4/80) antibodies were purchased from Protetintech Group, Inc. (Rosemont, United States).

Oil red O staining was conducted using a modified oil red O staining Kit (Solarbio, China; Cat # G1263). To begin, cells were fixed with 4% paraformaldehyde and then covered with oil red O buffer for a duration of 5 min. Subsequently, the cells were stained at RT for 15 min by adding the oil red O staining solution. Following this, the cells were treated with oil red O differentiation solution and stained with drops of mayer hematoxylin solution. After thorough washing, the stained fat droplets within the cells were observed and photographed using a light microscope. Positively stained areas were analyzed using ImageJ and normalized by controls.

An Oil Red O staining kit (G1262; Solarbio, Beijing, China) was used. Briefly, cells were washed with PBS, fixed with cytochrome fixative for 30 min, washed with 60% isopropanol, stained with Oil Red O staining solution for 10–15 min, and counterstained with Mayer hematoxylin solution for 1–2 min. Finally, cell images were collected under an inverted microscope (IX-73; Olympus, Tokyo, Japan) to observe the lipid droplets.
The triglyceride content was determined using an unkude triglyceride determination kit (Applygen Technologies, Beijing, China). The cells were washed three times with PBS and lysed in lysis buffer (R0010; Solarbio, Beijing, China). TAG content was determined via enzymatic colorimetry at 570 nm using a microplate reader (iMark; Bio-Rad, Hercules, CA, USA).

The HepG2 cells were seeded in 6-well plates and stained with a commercial Oil Red O staining kit (Solarbio, China). The HepG2 cells were washed with phosphate buffered saline (PBS) and fixed with ORO Fixative for 1 h. Cells were rinsed with ddH₂O, dipped in 60% isopropanol for 5 min, stained with the ORO stain for 20 min, and rinsed with ddH₂O 2–5 times to remove excess staining solution. The nuclei were stained with Mayer hematoxylin staining solution for 1–2 min and washed 2–5 times. ORO buffer was added for 1 min and finally, distilled water was added to cover the cells, which were observed under a microscope (Bio-Rad, USA). The Oil Red O staining was quantified based on the OD value at 490 nm using a microplate reader (Bio-Rad, USA).