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| How to Use BBB Kit™ ( RBE-12 ) |
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| Thawing (De-freezing) and Activation of BBB Kit |
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| RBE-12 (PET-12) |
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| [ PDF-2.1MB ] |
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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 |
630 μL |
500 μL |
500 μL |
| Brain-side |
1000 μL |
1500 μL |
1500 μL |
* Recipe: medium 2 10% PDS / DMEM F-12
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Dulbecco?fs Modified Eagle?fs 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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| thawing*1〜8 →medium 1*9〜10 →incubation*11→ medium 2*12〜14 →incubation*15 →experiment*16 |
| 1. |
Warm thawing-sol to 37 ℃, prior to De-freezing and warm up BBB Kit ™.
(Move frozen thawing-sol to 37 ℃ water-bath.) |
| 2. |
Move thawing-sol to clean-bench. |
| 3. |
Move a BBB 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 thawing-sol 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 630 μL thawing-sol to Blood-side (inside of Insert) (to all 12-wells).
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| 7. |
Stir up gently Blood-side (inside of Insert) with a pipette, 5 to 10 times.
Wipe up humidity on surface and bottom of BBB Kit ™.
(Do not stir up Brain-side.) |
| 8. |
Incubate BBB Kit ™ for 2 to 3 hrs in CO2 incubator. During this incubation, warm medium 1 to 37 ℃ |
| 9. |
See cells with inverted microscope.
Endothelial cells on Polycarbonate membrane of BBB Kit ™ (PC-12) cannot be seen by microscope, therefore microscopic examination for astrocytes on bottom side of lower compartment has to be done to check cell-proliferation.
RBE-12
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| endothelial cells (high magnification) |
astrocytes (high magni) |
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| 10. |
Remove thawing-sol from Brain-side, and add 1,500 μL medium 1 (red arrow), then remove thawing-sol from Blood-side, and add 500 μL medium 1.(Do not touch cells, carelessly. Add medium 1, very gently.)
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| 11. |
Incubate BBB Kit ™ with medium 1 CO2 incubator, overnight. |
| [ On day 1 ( the next day after thawing of BBB Kit ™ ) ] |
| 12. |
See astrocytes through polycarbonate membranes with inverted microscope.
RBE-12
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| endothelial cells (low magnification) |
astrocytes (low magni) |
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| 13. |
Warm medium 2 to 37 ℃ in water-bath.(Move frozen medium 2 to 37 ℃ water-bath. ) |
| 14. |
Remove medium 1 from Brain-side, and add 1,500 μL medium 2 (red arrow), then remove medium 1 from Blood-side, and add 500 μL medium 2 (Do not touch cells, carelessly. Add medium 2 very gently.)
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| 15. |
Incubate BBB Kit ™ with medium 2 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 ™ (RBE-12) |
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TEER in BBB Kit ™ (RBE-12) reaches more than 150 Ω×cm2, and maintains a plateau up to 7 days.
(Thawing-sol and medium 1, 2 do not contain cAMP and its analogs.)
*BBB Kit can be used from Day 4 to Day 6 after thawing. |
BBB Kit ™ (RBE12)
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MBT-24H
