Nov 23, 2022

Public workspaceEvaluating endocytic rate in cells.

DOI
dx.doi.org/10.17504/protocols.io.8epv5jjedl1b/v1
  • 1KU Leuven
Marine Houdou
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DOI: dx.doi.org/10.17504/protocols.io.8epv5jjedl1b/v1
External link: https://www.thermofisher.com/order/catalog/product/T23366?gclid=CjwKCAjwzY2bBhB6EiwAPpUpZkuhTJcss8FLP963Hi9idgxNuMgwOubQVDObHI_Ru5cHPICunDbHdxoC24IQAvD_BwE&ef_id=CjwKCAjwzY2bBhB6EiwAPpUpZkuhTJcss8FLP963Hi9idgxNuMgwOubQVDObHI_Ru5cHPICunDbHdxoC24IQAvD_BwE:G:s&s_kwcid=AL!3652!3!447292198772!!!g!!!10506731179!109642167771&cid=bid_pca_iva_r01_co_cp1359_pjt0000_bid00000_0se_gaw_dy_pur_con
Protocol Citation: Marine Houdou, Peter Vangheluwe, Nathalie Jacobs 2022. Evaluating endocytic rate in cells.. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5jjedl1b/v1
Manuscript citation:
Houdou M, Jacobs N, Coene J, Azfar M, Vanhoutte R, Haute CVd, Eggermont J, Daniëls V, Verhelst SHL, Vangheluwe P, Novel Green Fluorescent Polyamines to Analyze ATP13A2 and ATP13A3 Activity in the Mammalian Polyamine Transport System. Biomolecules 13(2). doi: 10.3390/biom13020337
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 03, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 72253
Keywords: ASAPCRN
Abstract
Assess endocytic rate in cells using tagged transferrin (Alexa647) and flow cytometry readout.
Guidelines
  • Make sure to treat the cells in medium without FBS.
Materials
0.25% Trypsin-EDTA: Gibco, 25200056

Albumin Fraction V: Carl Roth, 8076.4

Alexa647-Transferrin: T23366, invitrogen

Dulbecco's Phosphate Buffered Saline modified without calcium chloride and magnesium chloride (DPBS (-/-)): Gibco, D8537

Endocytosis inhibitors
  • Dynasore: D7693, Sigma
  • Genistein: ab120112, Abcam
  • Pitstop 2: SML1169, Sigma

TrypLE Express Enzyme: Gibco, 12604021

Versene Solution: Gibco, 15040
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.

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 ofAmount500 µL /well), for Duration00:30:00 at 37°C, 5% CO2 . The different conditions to consider are:


30m
No pre-treatment for:
  • blank samples
  • Alexa647-transferrin-only samples.

In this case, change cell culture medium to cell culture medium without FBS.
Pre-treatment with endocytosis inhibitors cocktail made of:
  • Amount100 µM Dynasore ,
  • Amount50 µM Genistein and
  • Amount50 µM Pitstop2 .
After pre-treatment, keep the plate on ice (4°C) for Duration00:15:00 .
15m
Add Alexa647-Transferrin at Amount50 µg /mL 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 Duration00:20:00 4°C (on ice)
20m
Incubate Duration00:20:00 37°C, 5% CO2 .
20m
Harvest cells and prepare samples for Flow Cytometry (FC).
Discard medium.
Wash with Amount500 µL /well Versene or DPBS (-/-).
Discard Versene or DPBS (-/-).
Add Amount200 µL /well 0.25 % Trypsin or TrypLE. Incubate Duration00:05:00 at RT .
5m
Add Amount500 µL /well 1 % BSA-DPBS (-/-), collect the cells and transfer in Eppendorf tubes.
CentrifugeDuration00:05:00 at 2500 rpm and 4°C.
5m
Discard supernatants and resuspend cell pellet in Amount250-500 µL 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.

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.