I found in the literature that nicotine, acting as an nAChR agonist, increases the proliferation of A549 cells (human lung adenocarcinoma cells). But when I treat A549 cells in 96-well plates with nicotine (concentration range from 0.1-5 uM) for different periods (from 1 to 3 days), I get no effect on cell proliferation (using alamarBlue assay, Neutral Red Uptake assay and microscopic observation). For experiments I am using DMEM (high glucose) cell medium supplemented with 10% FBS. Does anyone have an explanation as to why I am not getting an increase in cell proliferation?
I would like to thank you in advance for any answers/comments.
Dear Veno!
You do not observe cell proliferation with nicotine because you are using inappropriate assessment methods.
For example,
Proliferation Assays
Bromodeoxyuridine (BrdU) labeling kits were obtained from Roche Biochemicals. Cells were plated in poly-D-lysine coated chamber slides at a density of 10,000 cells per well and rendered quiescent by serum starvation for 36 hours. Cells were then re-stimulated with 1µM nicotine or 10% FBS for 18 hours. S-phase cells were visualized by microscopy and quantitated by counting 3 fields of 100 cells in quadruplicate. Data is presented as the percentage of BrdU positive cells out of the 100 cells counted.
Article Nicotine Induces Cell Proliferation, Invasion and Epithelial...
Figure 1
Nicotine can promote proliferation, invasion and survival pathways in A549 NSCLC cells. (A) Nicotine can induce S-phase entry in A549 NSCLC cells in a dose-dependent manner, as measured by BrdU assays. (B) Nicotine can induce anchorage-independent growth of A549 cells in soft-agar assay. Nicotine increased the size of the individual colonies but had little effect on the total number of colonies. (C) Nicotine can promote migration and invasion of A549 cells in Boyden Chamber assays, in a dose dependent manner, the maximal effect being observed at 1µM nicotine. (D) Nicotine can confer resistance to anoikis of SAECs. SAECs were treated with 1µM nicotine and plated on polyhema coated slides. Apoptosis was measured by TUNEL assays. (E) Treatment of A549 cells with 1µM nicotine caused morphological changes, similar to VEGF on 3D collagen gel assays.
I agree you used wrong method to assess proliferation
use brdu assay as recommended
also take care and attention about passage number
is your culture old?! it might effect proliferation
Passage numbers should be recorded on the flask and not allowed to reach high numbers. Cells undergo genetic drift and other genotypic variations with high passage number. The end result will be slow growing cells, hard to transfect cells and data sets that cannot be repeated due to the changing genetics of the cell.
- Badges
- Science topic
- Similar topics
- Clinical Pharmacology
- Dosing