Transwell® Permeable Supports: 25 Tips & Techniques

Practical Tips and Techniques for Transwell® Permeable Supports

Corning® Transwell® Permeable Supports have been a trusted laboratory standard for decades. During this time, researchers have refined many practical tips and techniques. As a result, these insights help improve consistency, reproducibility, and assay performance. 

Below are 25 proven techniques to help you get the most from Transwell® Permeable Supports.

Media Handling with Transwell® Permeable Supports

1. Always aspirate media from the basolateral side first during drug transport assays. Then remove media from the apical side.
2. When replacing media, reverse the order for improved consistency. Add media to the apical side first, followed by the basolateral side. 
3. For 24-well inserts, use alternating pipette tips on a multichannel pipettor. This approach simplifies liquid handling and reduces spills. 
4. Before TEER measurements, allow plates to equilibrate to room temperature. Consequently, you achieve more consistent readings. 
   

Cell Seeding Strategies for Transwell® Permeable Assays

1. When seeding cells, optimisation is critical for each cell type. Therefore, avoid assuming higher seeding density improves outcomes.
2. For migration assays, fine-tune seeding concentrations based on assay goals. Refer to Corning® guidance for chemotaxis optimisation.  
3. To seed cells on the insert undersides, invert the full plate onto the lid. Then add one central cell droplet per insert.
4. After cell attachment, reassemble and invert the plate again carefully. This method improves membrane coverage. 
5. Avoid seeding cells from cultures above 90% confluence. Instead, seed from cultures at 85-90% confluence. 
6. Pre-soak inserts with serum-containing medium to encourage faster attachment. This step often improves early cell adherence. 
   
   

Imaging and Visualisation Using Transwell® Permeable Supports

1. Polyester inserts deliver superior optical clarity during imaging. Therefore, they are ideal for visualisation studies. 
2. For polycarbonate membranes, fix and stain cells before imaging. Suitable stains include crystal violet, Giemsa, and haematoxylin.
3. Crystal violet also works as a simple fixative and stain. It effectively supports migration enumeration.
4. Always inspect the insert undersides for trapped bubbles after chemoattractant addition. Otherwise, bubbles may interfere with cell growth. 
   
   

Migration and Invasion Assay Techniques with Transwell® Permeable Systems

1. Swab non-migrated cells before fixation when using staining methods. This technique improves migration accuracy.
2. Alternatively, collect migrated cells using a dissociation solution. Then quantify cells using fluorescence detection. 
3. FlouroBlok™ membranes eliminate swabbing entirely. Consequently, they simplify migration and invasion workflows. 
4. Always verify FluoroBlok™ results visually using fluorescence microscopy. This step ensures data reliability. 
   
   

Plate Compatibility and Handling for Transwell® Permeable Supports

1. Use treated receiver plates when required, as 96 HTS plates ship untreated. Clear, white, and black formats are available. 
2. The 96 HTS Transwell® inserts include access ports near column one. These ports allow reservoir access without insert removal. 
3. Always pair FluoroBlok™ inserts with the correct Falcon® receiver plates. Proper plat alignment is essential for accurate results. 
4. Transport seeded inserts carefully to avoid medium transfer. Split media may carry cells into the receiver wells. 
   

Optimisation Insights for Transwell® Permeable Assays

1. Passage number significantly affects drug transport results. For MDCK cells, passages 11-25 typically perform best. 
2. Endothelial cells respond to growth factors in a bell-curve pattern. Therefore, optimise VEGF concentration carefully. 
3. Cell source selection also matters, including HUVEC versus HMVEC. Protein expression levels may vary between sources. 
   
   

Understanding FluoroBlok™ Performance in Transwell™ Permeable Supports

The FluoroBlok™ membrane shows minimal autofluorescence from 400-700 nm. However, bottom-reading modes may show background from the plate itself. 

Excessive gain or missing controls can misrepresent membrane performance. 

Need Expert Advice on Your Transwell® Permeable Assays?

Our scientific specialists are ready to help optimise your Transwell® Permeable Supports workflows. Contact Eliot, our Life Sciences Specialist, for personalised technical guidance today. 

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