1. Noggin siRNA Alkaline Phosphatase Pilot Study
1/5/15
Plate Layout24 Wells
Fibrinogen [Day1, 6 Hours]
- 48 Well Plate
- 70 mg Fibrinogen
- 350 uL RNase-freeH2O
- 63.3 uL of 250U/mL Thrombin
- 63.3 uL of 5.5 mg/mLCaCL2
- Sterile PBS
1. Warm the fibrinogen [1Hour]
2. Sterilelyprepare 350 uL of 200 mg/mL fibrinogensolution[25uL/well]bymixing 70mg
Fibrinogenand 350 uL of sterile RNase-free H2Ointhe hood [Stir,do not pipet]
3. Place the fibrinogensolutionin37C waterbath until dissolvedfully[1-2Hours]
4. Add25 uL/well of fibrinogensolutiontothe bottomof the well ina48-well plate
5. Prepare 63.3 uL of CaCl2by mixing348.15 ug of Cacl2 with63.3 uL of DI water. Filter.
6. Prepare 950 uL of ThrombinWorkingSolution[75uL/well]bymixing 823.3 uL Sterile PBS, 63.3
uL of thrombin, 63.3 uL of CaCl2 [13:1:1][15 min before use]
7. Add75 uL/well of ThrombinWorkingSolution,quicklymix withpipettiptoevencoatthe well
8. Incubate [4 Hours]
9. CoverwithParafilmandplace in4C fridge
Cells[Day2, 5 Hours]
- TransfectionMedia(AlphaMem, 10% FBS)
- MC3T3 Cells(240,000)
1. Warm the plate fromthe previousdaytoRT [30 min]
2. Passage cellsbutplace cellsintransfectionmediainsteadof culture media. Need 240,000 cells
at a concentrationof 10,000 cells/125 uL
3. Pre-wetwith200 uL of PBS[15 min]
4. Remove PBSandadd cells [1 well at a time]
- Notreatment(MC3T3 cellsinregularmedia)_
- NoFibrin/Treatment+Tryp
- BMP-2 treatment(10ug/mLBMP-2)
- BMP-2 No Fibrin/Treatment+Tryp

2. 5. Incubate [4 Hours]
6. Warm rhBMP-2 and prepare 2500 uL of 10 ug/mLof rhBMP-2 dilutingwithculture media
7. Remove transfectionmediaandadd100 uL of culture media+10ug/mL BMP-2 to eachwell
8. Incubate
Alkaline Phosphatase[Day5, 1 Hour]
- PBS
- 1% Triton
- AlkPhosKitTablets
- 600 uL 3 MNaOH
1. Remove mediaandrinse with200 uL of PBS ineachwell
2. Add__ uL of __ Trypsinto eachwell [Incubate 5 min]
3. Add__ uL of mediatoeach well
4. Transfereach well sample toa1.5 mL conical tube,centrifuge 5min@ 1500 rpm
5. Remove mediaandreconstitutewithPBS
6. Centrifuge,repeatuntil solutionisclear
7. Remove PBSandadd 100 uL of 1% Triton
8. Remove 50 uL of solutionfromeachconical tube andplace ina 96 well plate inthe original
arrangement
9. Remove 1 goldand silverfoil tabletfromfreezerandwarmto RT
10. Vortex tabletsinDIwateruntil dissolved [5mL per tabletpair]
11. Add100 uL of pNPPsolutiontoeachwell
12. Incubate at RT [30 min]
13. Add25 uL/well of 3 M NaOH (Need600 uL)
14. Readthe plate at405 nm