Experiment Sharing: Operation Steps Of Blood Brain Barrier Experiment

From The End Of The 19th Century, Paul Ellich Found This Barrier In An Experiment. Later Scientists Followed Up And Found That The Blood Brain Barrier (BBB) Played An Important Role In The Body. The Integrity Of BBB Structure And Function Is Of Great Significance For Maintaining The Function Of Neurons And Protecting The Central Nervous System From Pathogens, Inflammatory Factors And Damage. The Occurrence And Development Of Ischemic Cerebrovascular Disease Are Closely Related To The Destruction Of BBB Structure And Function. 1、   Preparation Of Experimental Reagents: 1; Prepare Calcium And Magnesium Free PBS Phosphate Buffer Solution. Cell Systems Human Brain Microvascular Endothelial Cells (product No.: ACBRI 376) Suggest Using Calcium And Magnesium Free Buffer Solution For Experiment. 2; By Diluting 0.5 Mg/mL Cellulose Solution And 0.3 Mg/mL Collagen IV Solution, Use 1x PBS To 100 Mg In 1.5 ML Microtubule Respectively/ μ L-aliquot, Preparation 10 μ G/mL Collagen IV And 10 μ G/mL Cellulose Solution. After That, Divide The Two Equal 100 μ L And 1800 μ L Of 1x PBS Was Mixed In 2 ML Microtubes And Stored At - 20 ° C. You Can Also Use The Special Coating Solution For Primary Cells Of Cell Systems (product Number: 4Z0-201), Wait For 2 Minutes After Planking, Suck Off The Coating Matrix, And Directly Lay The Cells, Which Saves Time And Is Simple And Convenient. 3.  Prepare The Culture Medium. The Classic Cell Systems Culture Medium (product Number: 4Z0-500) Contains The Coating Solution And Growth Factors, And Does Not Contain Antibiotics. 4.  To Prepare Trypsin, Dilute 5 ML Of 45 ML Of 1x PBS 10x Concentrated Trypsin EDTA Solution In A 50 ML Tube, Prepare 1x Trypsin EDTA Solution And Store It At 4 ° C, Or Cell Systems Generation Kit (product Number: 4Z0-800), Including PBS, Trypsin, And Trypsin Neutralization Solution, To Solve The Preparation Problem Of All Digestion Reagents In One Set. 5.  Weigh 10 G LB Broth In A 500 ML Glass Bottle To Prepare 500 ML LB Medium. Add 500mL Of Disinfected Water And Autoclave. 6.  Weigh 10 G LB Broth And 7.5 G Agar In A 500 ML Glass Bottle, And Prepare LB Agar. Before Autoclaving, Add 500mL Of Sterilizing Water And Do Not Close The Cap Of The Flask. Autoclave And Allow Solution To Cool Until Accessible& Nbsp; 2、 Growth Of Blood Brain Barrier Cells 1; When Using A 12 Hole Plate, Please Seed The Cell Coating Solution Into The Hole. 2.  Unzip The Board And Each Blade In The Biosafety Cabinet, And Perform Further Steps There. Use The Sterilizing Forceps To Grasp The Blade And Move It On Its Wide Base. 3.  Use 90 μ 10 μ G/mL Collagen IV And 10 μ The G/mL Fibrin Mixture Covers The Porous Membrane Of Each Blade. After That, They Were Incubated In The Cell Incubator For 24 Hours And Incubated At 37 ° C. 4.  Transfer 1 ML Of 1x PBS Into Each Blade, And Then Suck Out The Solution With A Vacuum Pump For Cell Culture. Wash The Blade Twice& Nbsp; Note: If The Cell Systems Cell Coating Solution Is Used, The First Three Steps Are: Use A Pipette To Move The Coating Solution Into The Pore Plate, And Suck It Out After 2 Minutes. Do Not Clean It, But Directly Perform The Fourth Step. 5.  The Membrane Was Balanced By Transferring 0.5 ML Of Preheated Cell Systems Classical Cell Culture Medium And 1.5 ML Of The Lower Chamber In The Upper Part. Incubate At 37 ° C For 30 Minutes In A Cell Incubator With 5% CO2 Atmosphere; Seed 2 X 105 Human Microvascular Endothelial Cells Entered Each Upper Chamber And Incubated 12 Well Plates In A Cell Incubator At 37 ° C. 7.  Use A Vacuum Pump To Suck Out The Culture Medium From The Cell Culture Medium, Then Wash The Monolayer With 10 ML Of 1x PBS, And Then Use The Vacuum Pump To Aspirate The Solution. 8.  Suck 5ml Of Medium Out Of The Culture Bottle, And Completely Cover The Cells With 1x Concentrated Trypsin EDTA. Incubate The Flask In The Cell Incubator At 37 ° C For 3-5 Minutes. Note: If The Cells Are Not Separated, Please Tap The Culture Bottle To Let The Cells Fall Off. 9.  Add Medium Containing Serum To Neutralize 10 At 210 X  G, Centrifugate The Suspension For 3 Minutes, Remove The Supernatant With A Vacuum Pump, And Resuspension The Cells In A 5 ML Medium With A Pipette. 11. & Nbsp; 10% Of The Cell Suspension μ L And 10 μ 0.4% Trypan Dye Of L Was Mixed In 1.5 ML Microtubule And Cell Counter Was Used. Will 10 μ The Mixture Of L Is Added To The Slider Of The Counting Chamber, Put Into The Cell Counter, And Then Start Counting. 12. . Focus The Counter On The Cell So That Its Edges Are Dark Blue And White In The Middle. After That, Start The Appropriate Cell Counting Program. 13. To Calculate The Volume Of Each Blade, Separate The Calculated Concentration Of Cell Suspension Of 2 X 10 ^ 5 Cells Obtained By Each Blade. 3. The Blood Brain Barrier Model Was Incubated At 37 ° C In 12 Well Plates For 14 Days. Change The Culture Medium Of The Upper Chamber Every Day; 0.5 ML, And Replace 1.5 ML Of Lower Chamber Medium Every 2-3 Days. Please Preheat The Medium Before Changing It. Vacuum Pump Inspires Air To Avoid Bubbles Note: Work Carefully To Avoid Contacting The Film. The Cells Were Imaged Under A Microscope To Check Their Status And Determine Their Confluence. Ensure The Convergence Is 100% After 14 Days. 4. Preparation Of Bacteria 1 On The Day Before The Measurement, The Coliform Bacteria Were Recovered In LB Medium. Incubate At 37 ° C For 24 Hours, And Incubate 180rpM2 In The Incubator Escherichia Coli Strains Were Cultured On LB Agar Plates At 4 ° C. Take A Picking Tube With Disinfection And Put The Picking Into The Culture Tube Prepared With 3 ML LB Agar Medium. 3. Prepare An LB Agar Plate For Each Blade And Fill The Petri Dish To Halve Its Total Volume. Let Them Become Solid And Store At 4 ° C. 5.   Cell Drug Experiment 1. On The 14th Day After Sowing, The Cells Were Treated With Compounds Or The Skin Transfer Resistance (TEER) Was Measured,. 2. If The Cells Are Treated With The Compound Of Interest, Dilute The Compound To The Adjusted Concentration And Dilute It To The Complete Medium. Add 0.5 ML Of The Mixture To The Upper Chamber And 1.5 ML To The Lower Chamber. Cultivate Again. After That, The Normal Cell Fluid Exchange Was Performed By Suction And Fluid Transfer Through The Vacuum Pump. 6. Permeability Measurement 1 In Order To Obtain A Constant Bacterial Concentration, The Optical Density Was Measured With A Photometer With A Wavelength Of 600 Nm. Use LB Medium To Dilute The Overnight Bacterial Solution In A 50 ML Falcon Tube To 0.5 ± 0.05 OD600. Working On Ice. 2. Add 1ml LB Medium To The Cuvette. Start The Photometer And Put The Cuvette In, With The Marked Side Facing Forward. Press "Blank" At The Bottom And Measure The Blank Value. 3. Inject The Bacterial Solution Into The Cuvette, Put It Into The Cuvette, And Then Press The Bottom Sample To Measure The Density Of The Bacterial Solution. Repeat The Measurement During Dilution Until The Final Concentration Is Obtained. 4. When Working In The Biosafety Cabinet, The Prepared 12 Hole Plate And OD600=0.5 Bacterial Solution Can Be Used To Treat Bacteria. Add Only 450 µ L Of Bacterial Solution To Each Upper Chamber Containing 0.5 ML Of Medium. 5. Incubate The 12-well Plate In An Incubator At 37 ° C For 6 Hours. 6. Use A Pipette To Take 50 µ L Culture Medium Samples From Each Lower Chamber By Removing The Inserts With Tweezers. Be Careful Not To Spill The Culture Medium From The Upper Chamber To The Lower Chamber. 7. Place Each Sample On A Separate Agar Plate. Drop The Sample On The Plate And Draw Out The Solution With A Cell Spreader. 8. Incubate The Agar Plate In The Incubator At 37 ° C For 24 Hours. 9. Calculate The Number Of Colonies In Each Plate; Cell Systems Blood Brain Barrier References:; Cysteinyl Leukotriene Receptor Type 1 Antagonist Montelukast Protects Against Injury Of Blood–brain Barrier" Zhou Et Al. Inflammopharmacology, 2019" Alterations Of The Endocannabinoid System In Post-ischemic Endothelial Cells Of The Blood Brain Barrier" Thurston (Dissertation) 2019Exosome-Mediated Transfer Of ACE2 (Angiotensin-Converting Enzyme 2) From Endothelial Progenitor Cells Promotes Survival And Function Of Endothelial Cell" Wang Et Al. Oxidative Medicine And Cellular Longevity, 2020" RAGE And CCR7 Mediate The Transmigration Of Zika-infected Monocytes Through The Blood-brain Barrier" Costa De Carvalho Et Al. Immunobiology, 2019" Binding Heterogeneity Of Plasmodium Falciparum To Engineered 3D Brain Microvessels Is Mediated By EPCR And ICAM-1" Bernabeu Et Al. MBio, 2019" In Vitro Cell Models Of The Human Blood-Brain Barrier: Demonstrating The Beneficial Influence Of Shear Stress On Brain Microvascular Endothelial Cell Phenotype" Rochfort And Cummins Et Al. Blood-Brain Barrier, 2018" Modulation Of Glucocorticoid Receptor In Human Epileptic Endothelial Cells Impacts Drug Biotransformation In An In Vitro Blood–brain Barrier Model" Ghosh Et Al. Epilepsia, 2018.