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Sample Protocol
Caco-2 Membranes in 6-Well SnapWell® Plates
Programmed protocols guide the operator in setting up the various steps in the procedure but can be modified to suit application and preference. Different tests and plate types may require different protocols.
Outlined below are steps for processing Corning #3407 6-well SnapWell® plates, including Coating, Conditioning, Seeding and Feeding.
1. STARTING PLATES
- Load new plates into incubator.
2. COATING
- Remove plate from incubator, transfer to Hood 1 and remove lid
- Move plate to coating station, release
- Coat plate, inner wells with 0.2 ml collagen
- Regrip plate and replace lid
- Allow plate to dry in Hood 1 tower for 4-5 hours
3. CONDITIONING
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Prepares plates for seeding and can be done after plates dry for 4-5 hours
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- Remove coated plate from Hood 1, transfer to Hood 2 and remove lid
- Place plate under camera, view/locate inserts
- Place plate under dispenser; dispense 2.0 ml SFM into outer wells
- Replace plate lid, transfer plate to incubator; incubate for 2 hours
- Remove plate from incubator, transfer to Hood 2 and remove lid
- Place plate in dispenser, dispense 0.5 ml of SFM into inner wells
- Replace plate lid, return plate to incubator; incubate for 1 hour
- Remove plate from incubator, transfer to Hood 2 and remove lid
- Place plate under camera, view/locate inserts
- Place plate under aspirator, aspirate media from outer wells
- Place plate under dispenser, dispense 2.0 ml MWS into outer wells
- Replace plate lid, place in incubator for at least 30 minutes.
- After last plate is conditioned begin seeding
4. SEEDING
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Cell suspension is maintained @ 37°C, then shaken gently with 5% CO2 to maintain proper pH and keep cells in suspension.
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- Remove plate from incubator, transfer to Hood 2 and remove lid
- Place plate under aspirator, aspirate from inner well
- Replace plate lid, transfer plate to Hood 1 and remove lid
- Place plate into seeding station, seed plate with 0.5 ml into inner well
- Replace plate lid, return plate to incubator; incubate for 48 hours
- Refeed (see feeding below)
5. FEEDING
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Feeding is done on the dispenser using MWS media with serum @ 37° C ±1.0°
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- Remove plate from incubator after 48-hour incubation
- Regrip plate, bring into Hood 2 and remove lid
- Place plate under camera to view/locate inserts
- Place plate under aspirator, aspirate media from outer wells, inner wells
- Move plate to dispenser; dispense 0.5 ml MWS into inner wells and 2.0 ml MWS into outer well
- Replace plate lid, return plate to incubator and incubate for 48 hours
- Refeed after 48 hours for a total of 21 days
- SFM – serum-free medium for conditioning
MWS – medium with serum for feeding
See a Cytogration system in action.
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