I'm currently doing total cholesterol assay in SH-SY5Y cells treated with 0, 25, 50, 100ug cholesterol/mL (using cholesterol:mbcd, sigma)
Lipid extraction is done by chloroform and methanol. After taking chloroform sublayer it is dried in N2 gas, dissolved again in isopropanol and then absorbance is measured in 500nm.
The problem is that mg TC/mg protein is too high (almost 100 times bigger) compared to preceding studies.
The mg TC/mg protein of control(untreated) SH-SY5Y measured was about 0.1656.
Its value is similar(or even higher) to animal tissue rather than cell.
Can anyone help me with this problem, please?
Dear Dr. Juhyun Chung
It sounds like you're encountering an unexpected discrepancy in the total cholesterol (TC) to protein ratio in your SH-SY5Y cell samples. Genreal Protocol are
After washing and incubation in a growth medium for 6 h, the cells were counted. A phase separation method (chloroform/methanol, 2:1) was used to extract cholesterol from 8.5 × 105 cells. The organic phase was evaporated, and the lipids were dissolved in 2-propanol. Cholesterol and cholesterol esters were measured as per the manufacturer's protocol (Biovision). The samples were diluted 1:25 in 50 μl of the cholesterol reaction buffer. An equal volume of reaction mix containing cholesterol reaction buffer, cholesterol probe, enzyme mix, and cholesterol esterase was added to the samples and incubated in the dark for 60 min at 37 °C. Absorbance was read at 562 nm using an ELx800uv microplate reader. The values obtained were subtracted from the blank containing the reaction buffer and extrapolated from the standard curve for cholesterol. Total cellular cholesterol was expressed as μg/μl per sample.
Here are some potential factors to consider and troubleshooting steps you can take
Extraction Efficiency: Review your lipid extraction protocol to ensure it efficiently extracts cholesterol from the cells. Variations in extraction efficiency could result in differences in measured TC levels.
Normalization: Confirm that your protein assay is accurately quantifying the protein content of your samples. Inaccurate protein quantification could skew the TC to protein ratio.
Validation: Validate your findings with complementary assays or techniques, such as thin-layer chromatography or mass spectrometry, to confirm the accuracy of your TC measurements.
By systematically evaluating these factors and troubleshooting any issues, you can hopefully identify the cause of the discrepancy in TC to protein ratio and ensure the reliability of your experimental results.
Article Lipid Analysis of the 6-Hydroxydopamine-Treated SH-SY5Y Cell...
Article Quercetin 3-Glucoside Protects Neuroblastoma (SH-SY5Y) Cells...
https://www.jstage.jst.go.jp/article/jat1994/2/2/2_2_117/_article
Hello Juhyun Chung
Cholesterol is an essential component of cell membranes. While it is not a storage form, a substantial amount of cholesterol is present in membranes. If you are using differentiated (even to some extent) SH-SY5Y cells, their membrane-to-volume ratio is much higher, so having a high cholesterol value is expected.
- Badges
- Science method
- Similar topics
- Mathematics
- Statistics
- Statistical Modeling
- Analyses of Variance
- ANOVA