Endocytosis and internalization assay in primary neuronal culture
Arpine Sokratian, andrew.west west
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Abstract
This protocol outlines a method for quantifying endocytosis in primary neuronal cultures, specifically focusing on different a-synuclein fibril structures. By utilizing pH-sensitive dye conjugated to experimental proteins/fibrils, we can identify endocytosis pathways and analyze intensity changes in the pHrodo-specific channel from real-time acquired images at various time points.
Steps
Preparation of endocytosis inhibitors
For the assay we use DIV7 primary hippocampal neuron culture plated in 48-well plates
Prepare stock solution of the endocytosis inhibitors in DMSO:
| A | B |
|---|---|
| 2.3 mM | Wortmannin |
| 50 mM | Dyngo |
| 10 mM | Pitstop 2 |
| 384 mM | methyl-β-cyclodextrin (MβCD) |
| 50 mM | ethyl-isopropyl amiloride |
Recommended concentrations for stock solutions (stocks can be freezed at -80C for couple of days
At the day of the experiment, thaw down the stock solutions in a water bath and dilute the drugs to reach concentrations:
| A | B | C | D | E | F | G |
|---|---|---|---|---|---|---|
| Ethyl-isopropyl amiloride (EIPA) | 50 uM | 500uM: 10 ul of stock + 990ul PBS | ||||
| Dyngo | 10 uM | 10 mM: 20ul of the drug + 80 ul (50% DMSO) | for 100 uM of Dyngo: 5 ul of the 10mM dyngo + 495 ul of PBS | |||
| Wortmannin | 0.2 uM | 10 uM: 4ul of the stock + 916 ul PBS | 1uM: 200 ul of 10 uM + 800 ul of PBS | |||
| Pitstop 2 | 15 uM | 150 uM: 15ul of P + 984 ul PBS | ||||
| Methyl-β-cyclodextrin (MβCD) | 2 mM | 20 mM: 25 ul of stock + 475 ul PBS |
Drugs are prepared to be diluted to final concentration in the cell culture
Before adding protein/drug of interest, add endocytosis inhibitors 30 minutes before the treatment. As calculated for 48-well plates with 3 mL of the media, 30 ul of diluted drugs are sufficient. ** Note: Be careful adding the diluted drugs (should be RT). Place the plate back to the cell culture incubator.
After 30 minutes, add the protein/drug of interest supplement with
As for phRodo-conjugated fibrils, please, see the protocol describing the conjugation process (step 5):
As a control for endocytosis add lysotracker
After two hours of incubation, start taking images of each well at the high-speed (high sensitive mode)
Continue acquiring images every two to four hours up to 48 hours of incubation.
Calculate the images using cell count (Hoechst) for normalization and calculate the signal for each time-point as a proportion to the maxima of the signal
Calculate the signal from Lysotracker after 30 minutes of incubation and compare the control (no endocytosis inhibitors) to the experimental reactions.
