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| How to Use BBB Kit™ ( MBT-24H ) |
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| Thawing (De-freezing) and Activation of BBB Kit™ |
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| MBT-24H |
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| MBT-24F |
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| [ PDF-1.28MB ] |
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| 1. Schedule after thawing BBB Kit™ |
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Amounts of solutions (to be added)
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Thawing-sol |
medium (1) |
medium (2) * |
| Blood-side |
200 μL |
300 μL |
300 μL |
| Brain-side |
1000 μL |
1200 μL |
1200 μL |
* Recipe: medium 10% PDS / DMEM F-12
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Dulbecco's Modified Eagle's Medium/ Nutrient Mixture F-12 Ham (DMEM F-12) |
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| Fetal Bovine Plasma Derived Serum (PDS) |
10% (v/v) |
| Heparin |
100 μg/mL |
| basic fibroblast growth factor (bFGF) |
1.5 ng/mL |
| Insulin-Transferrin-sodium Selenite (ITS) |
insulin 5 μg/mL,
transferrin 5 μg/mL,
sodium selenite 5 ng/mL |
| Hydrocortisone |
500 nM |
| Gentamicin |
50 μg/mL |
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| 2. Storing of BBB Kit™, thawing-sol and incubation medium |
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BBB Kit™ is stored at -80 ℃, and can be used within one month.
Thawing-sol. and incubation medium are stored at below -20℃. |
| 3. Protocol (procedure of activating BBB Kit™) |
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| 1. |
Warm Medium to 37 ℃, prior to De-freezing and warm up BBB Kit ™.
(Move frozen thawing-sol to 37 ℃ water-bath.) |
| 2. |
Move Medium to clean-bench. |
| 3. |
Move a BB Kit™ in frozen to clean-bench. Take off seals.
(Do not take a minute.) |
| 4. |
Wipe up waterdrops (humidity) on BBB Kit™ with clean papers. |
| 5. |
Add 1,000 μl Medium to Brain-side (to all 12-wells),
through an opening between Inserts.
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Do not touch membrane of insert with pipette, and do not move insert, during procedures of
#1 to #5. |
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| 6. |
Add 200 μl Medium to Blood-side (inside of Insert) (to all 12-wells).
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| 7. |
Wipe up humidity on surface and bottom of BB Kit™. |
| 8. |
Incubate BBB Kit™ for 2 to 3 hrs in CO2 incubator. During this incubation, Medium warm to 37 ℃ |
| 9. |
See cells with inverted microscope.
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| astrocytes |
enodthelium |
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Endothelial cells on Polyethylene terephthalate membrane of BBB Kit™ (MBT-24) cannot be seen by microscope, therefore microscopic examination for astrocytes on bottom side of lower compartment has to be done to check cell-proliferation. |
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| 10. |
Using a pincet to lift-up the insert, remove the present Medium of the brain-side. Return the insert to the plate, then remove the present Medium of the blood-side. Add 300 μL new medium to the blood-side very gently, and then 1200 μL new to the brain-side.
Notice that do not touch the cells or membrane of the insert.
Repeat this work to finish all 12 wells.
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| 11. |
Incubate BBB Kit™ with Medium in CO2 incubator, overnight. |
| [ On day 1 ( the next day after thawing of BBB Kit™ ) ] |
| 12. |
See astrocytes through polyethylene telephtalate membranes with inverted microscope.
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| astrocytes |
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| 13. |
Warm Medium to 37 ℃ in water-bath. (Move frozen Medium 37 ℃ water-bath. ) |
| 14. |
Remove Medium from Brain-side, and add 900μL ( (red arrow), then remove from Blood-side, and add 200μL (Do not touch cells, carelessly. Add Medium very gently.)
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| 15. |
Incubate BBB Kit ™ with Medium in CO2 incubator for 3 days. (from thawing day (Day 0) to Day 4) |
| 16. |
On Day 4, BBB Kit™ is activated functionally, and maintains BBB function until Day 7. Use activated-BBB Kit™ on Day 4. (You can store activated- BBB Kit ™ in CO2 incubator at 37 ℃. We recommend you to use the BBB Kit ™ until Day 6.) |
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| 4. TEER (trans endothelial electrical resistance ) in BBB Kit ™ (MBT-24H) |
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TEER in BBB Kit ™ (MBT-24) reaches more than 150 Ω×cm2, and maintains a plateau up to 6 days.
(Medium does not contain cAMP and its analogs.)
*BBB Kit™ can be used from Day 4 to Day 6 after thawing. |
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【Equipment】
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MBT-24H
