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How to Use BBB Kit™ ( RBE-12 )

Thawing (De-freezing) and Activation of BBB Kit

RBE-12 (PET-12)
RBE-12 Protocol
[ PDF-2.1MB ]

1. Schedule after thawing BBB Kit ™

  Amounts of solutions (to be added)
  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
  Dulbecco?fs Modified Eagle?fs Medium/ Nutrient Mixture F-12 Ham (DMEM F-12)  
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

2. Storing of BBB Kit ™, thawing-sol and incubation medium
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 ™)
thawing*1〜8medium 1*9〜10 →incubation*11→ medium 2*12〜14 →incubation*15 →experiment*16

  [ On thawing (day 0) ]

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.
Do not touch membrane of insert with pipette, and do not move insert, during procedures of #1 to #5.
6. Add 630 μL thawing-sol  to Blood-side (inside of Insert) (to all 12-wells).
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.

endothelial cells (high magnification) astrocytes (high magni)
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.)
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.

endothelial cells (low magnification) astrocytes (low magni)
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.)
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.)

Schedule (example)

4. TEER (trans endothelial electrical resistance) in BBB Kit ™ (RBE-12)
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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