Co-Culture in Millicell Cell Culture Inserts Protocol

Coat T-75 flasks with 10 mL of 0.1% gelatin in DPBS and incubate for at least 30 minutes at 37°C.
Thaw PMEF vial (s) quickly in a 37°C water bath, transfer to 15 mL tube already containing 10 mL warm medium. Gently invert tube, and pellet cells at 4°C, ~1000 rpm for approximately 4–5 minutes.
Remove supernatant, resuspend cells, remove gelatin from plates/flasks, and aliquote feeder cell suspension per densities recommended below.
Remove excess gelatin from flask prior to seeding.
Seed flask with MEF feeder cell suspension: approximately 1.5 × 105 cells per mL MEF should result in 95% confluence within 24 hours.
Incubate at 37°C overnight.

Coat T-75 flasks with 10 mL of 0.1% gelatin in DPBS and incubate for at least 30 minutes at 37°C.
Thaw PMEF vial (s) quickly in a 37°C water bath, transfer to 15 mL tube already containing 10 mL warm medium. Gently invert tube, and pellet cells at 4°C, ~1000 rpm for approximately 4–5 minutes.
Remove supernatant, resuspend cells, remove gelatin from plates/flasks, and aliquote feeder cell suspension per densities recommended below.
Remove excess gelatin from flask prior to seeding.
Seed flask with MEF feeder cell suspension: approximately 1.5 × 105 cells per mL MEF should result in 95% confluence within 24 hours.
Incubate at 37°C overnight.

Feed ESC on MEF feeder layer with fresh ESC media or pass cells, at a 1:2 ratio, if required. (After thawing ESC, 2–3 passages are preferred before seeding onto a Millicell-24 or Millicell 96-well plate. Both cell types are lifted at once and passed on to a new T-75 containing inactivated MEF.) ESC colonies grown on Millicell-96 cell culture insert 1.0 PET membrane, stained for alkaline phosphatase activity, after culturing via indirect co-culture with ESC in apical well, at a 200 cell per well seeding density, and MEF in the single-well feeder tray.

Feed ESC on MEF feeder layer with fresh ESC media.
Coat Millicell-24 or Millicell-96 single-well feeder tray with approximately 5–10 mL of 25 µg/mL fibronectin in DPBS and incubate for 45 minutes at room temperature.
Remove excess fibronectin from single-well tray.
Thaw MEF using protocol from Day 1, section A.
Seed fibronectin coated single-well tray with MEF cell suspension: approximately 1.67 × 106 MEF cells per single-well tray will result in 95% confluence within 24 hours.
Cover with lid and incubate single-well tray at 37°C overnight.

Lift ESC and MEF feeder cells from T-75 flasks:
1. Wash flasks 2X with 10 mL of pre-warmed DPBS (incubate 1–2 minutes per wash).
2. Remove DPBS and add 3 mL TrypLE and incubate at room temp for 2–3 minutes.
3. Monitor detachment of cells with an inverted microscope and add ESC media to inactivate TrypLE.
4. Mix well and wash flask wall to remove all cells from flask.
Separate ESC from MEF feeder cells:
1. Transfer ESC/MEF cell suspension to a new T-75 flask and incubate at 37°C for 45 minutes.
2. Remove non-adherent cells (ESC) and transfer to another new T-75 flask and incubate at 37°C for 45 minutes.
3. Remove non-adherent cells (ESC) again and seed cell culture filter plate wells with ESC suspension.
Seed apical wells of the cell culture plates with ESC. Seed approximately 200–500 cells per well in 100 µL ESC media for Millicell-96 plates
and approximately 1000–1500 cells per well in 400 µL ESC media for Millicell-24 plates.
Remove media from the MEF seeded single-well trays and replace with approximately 28–32 mL ESC media.
Combine ESC seeded cell culture filter plates to MEF seeded single-well trays.
Incubate assembly at 37°C overnight.

Analyze alkaline phosphatase activity to demonstrate that ESC is undifferentiated with an alkaline phosphatase detection kit.

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