To study the initial interactions of EVs with cells, this protocol would be a good starting point. Here cells are not labelled and only EVs are labelled

Requirements:

Imaging buffer

Imaging buffer must be free from phenol red as it interferes with imaging. Depending on cell types imaging buffer can be

• 1X PBS,

• Hepes buffer DMEM,

• Live cell imaging buffer e.g

https://www.thermofisher.com/order/catalog/product/A14291DJ#/A14291DJ

EVs can be stained with membrane stains such as DIL, DIO, DiD, PKH26 or PKH67. Recommendation would be to use covalent linked dyes like exoglow protein label to maintain the identity of EVs once internalized by the cells.  

• As a starting concentration, 10 nm to 100nm would be a good starting concentration for EVs. Use the concentration recommended by the supplier. For DIL, Did and DIO check this link https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2Fmp22885.pdf&title=VnlicmFudCBDZWxsLUxhYmVsaW5nIFNvbHV0aW9ucw==

• Filter out the free dye before adding labelled EVs to the cells. This is can be achieved using a Molecular cut off filter, Ultracentrifugation or dialysis.
  
  A nice summary is described of different methods in this article

https://www.sciencedirect.com/science/article/pii/S0005273618300932.

Plating cells

Plate cells overnight to reach confluency of 60-70% next day.

• For 8 well Ibidi chamber plate 8K cells per well

• for 18 well Ibidi chamber plate 2K cells

This is a rough guideline for large cells like keratinocytes and for smaller cells like HEK, RAW and J774 increase the numbers by 5 times.

Imaging of EVs with cells

2). Before imaging wash cells gently three times with warm (37C) imaging buffer.

3). Add EVs to a final concentration of 10⁸ – 10¹⁰ to cells.

1. For 8 well Ibidi chambers use 200ul imaging solution
   
2. For 18 well ibidi chamber use 40ul imaging solution.