Evaluating endocytic rate in cells.

Marine Houdou, Nathalie Jacobs, Peter Vangheluwe

Published: 2022-11-24 DOI: 10.17504/protocols.io.8epv5jjedl1b/v1

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Abstract

Assess endocytic rate in cells using tagged transferrin (Alexa647) and flow cytometry readout.

Before start

Prepare cell culture medium without FBS and keep it at 37°C.

Prepare Flow Cytometry (FC) buffer made of 1% Albumin Fraction V in DPBS (-/-).
Prepare Eppendorf tubes and FC tubes by labeling them and keeping them on ice.

Steps

Seed cells in 12 wellplate that they reach 70-80% confluency the day of the experiment.

Note

This protocol is made for a 12 wellplate format. Adaptations need to be done to scale up or down.

The day of the experiment, remove cell culture medium and pre-treat the cells in medium without FBS in a final volume of500µL, for 0h 30m 0s. The different conditions to consider are:

2.1.

No pre-treatment for:

blank samples
Alexa647-transferrin-only samples.

In this case, change cell culture medium to cell culture medium without FBS.

2.2.

Pre-treatment with endocytosis inhibitors cocktail made of:

100µM,
50µM and
50µM.

2.3.

After pre-treatment, keep the plate on ice (4°C) for 0h 15m 0s .

Add Alexa647-Transferrin at 50µg in each well, except for the blank samples .

Note

Alexa647-Transferrin is added to each well containing 500µL of either culture medium withtout FBS or pre-treated medium including endocytosis inhibitors in culture medium without FBS.

Incubate 0h 20m 0s

Incubate 0h 20m 0s.

Harvest cells and prepare samples for Flow Cytometry (FC).

6.1.

Discard medium.

6.2.

Wash with 500µL Versene or DPBS (-/-).

6.3.

Discard Versene or DPBS (-/-).

6.4.

Add 200µL 0.25 % Trypsin or TrypLE. Incubate 0h 5m 0s.

6.5.

Add 500µL 1 % BSA-DPBS (-/-), collect the cells and transfer in Eppendorf tubes.

6.6.

Centrifuge0h 5m 0s at 2500 rpm and 4°C.

6.7.

Discard supernatants and resuspend cell pellet in 250-500µL µL 1 % Albumin Fraction V in DPBS (-/-) , depending on the size of the cell pellet.

Note

Samples can't be too concentrated or too diluted to be ran on a flow cytometer. If samples are too concetrated, cells will form clumps, they won't be single cells anymore and this will damage the flow cytometer.If samples are too diluted, acquisition at the flow cytometer will take very long time.

6.8.

Filter cell suspension through Nylon filter into FC tubes.

Keep on ice until acquisition at the flow cytometer. Record 10,000 live events per sample.

Evaluating endocytic rate in cells.

Abstract

Before start

Steps

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