How To Do Endodermis Tube? The Most Complete Operation Steps Are Coming

The New Blood Vessels In The Body Are Composed Of Three Processes: Angiogenesis, Arteriogenesis And Angiogenesis. Arteriogenesis Involves Remodeling Of Existing Blood Vessels. Maturation And Production Of Fully Developed Functional Arteries Depend On The Continuity Of Endothelial Cell Lining And Basement Layer. In Addition, It Is Also Related To The Size And Shape Of Endothelial Cells, Intersections, And The Number Of Plasma Membrane Bodies. Functional Heterogeneity Between Endothelial Cells Is Involved In Controlling Vascular Contraction And Expansion, Blood, Coagulation, Fibrinolysis, And The Presence Of Antigens Atherosclerosis And Catabolic Lipoproteins& Nbsp; But, How Are Blood Vessels Generated? Master The Following Points To Easily Complete The Angiogenesis Experiment: 1; Prepare P3-P5 HUVEC Cells, ECGM Endothelial Cell Culture Medium, Matrix Glue, Phosphate Buffered Saline, And Culture Consumables 2; Preparation Of Conditioned Medium From Target Cells 3; To Prepare Conditioned Medium (CM), Seed Target Cells And Grow To 30-40% Confluence (depending On The Growth Rate Of The Cell Line), Replace The Growth Medium With Serum-free ECGM (for Example, 10ml For T75 Tissue Culture Bottle; It Does Not Matter When There Is Or Does Not Exist Antibiotics) For 24 Hours, And Then Harvest CM When The Cells Reach 60-80% Confluence In T75 Tissue Culture Bottle. 4.  Make 0.5ml Of CM Aliquot And Store It At - 80 ° C, If It Is Not Used Immediately After Collecting CM& Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; & Nbsp; HUVEC (Qida Article No.: CD0290, P2) 5; Before The Tube Formation Test, Remove The Serum Double Antibody To Starve HUVEC Cells For 3-6 Hours. If T25 Cell Culture Bottle Is Used, Add About 5ml Of Basic Medium For Culture. 6. After The 96 Well Plate Is Coated With The Diluted Matrigel, The Cells Are Digested With 0.25% EDTA Trypsin. After The Cells Fall Off, They Are Neutralized With Double ECGM Medium Containing Serum, And Centrifuged At 1200 Rpm (276 Rpm) At Room Temperature × G) 3-4 Min, Resuspension In 2-3ml Serum Free ECGM, Measure The Concentration Of HUVEC By Counting Cells, And Blow Up And Down Several Times To 4 × 10 ^ 5 Cells/ml Use ECGM Without Serum To Resuspension HUVEC Cells For Times To Ensure Uniform Single Cell Suspension Note: For Diluted Matrix, The Thawing/freezing Cycle Should Be Reduced As Much As Possible. 7. Mix Well, And Transfer 500 μ L HUVEC Cell Suspension Into A 1.5ml Tube At The Same Time. Use A Desktop Centrifuge To Rotate Cells At 4000rpm (1100rcf) For 3 Minutes. Carefully Suck Out The Supernatant And Do Not Touch The Cell Precipitation. Remove As Much Supernatant As Possible. Prepare An Appropriate Number Of HUVEC Cells In 1.5 ML Tubes According To The Number Of Target Cells To Be Used. Thaw 0.5ml Aliquots Of CM From The Target Cell Line Collected In Step 2 Of Procedure A, And Supplement With Fetal Bovine Serum To The Final Concentration Of 1% To Resuspension HUVEC Cell Precipitation In Step 5 Of Procedure B. Note: Each Target Cell Line Should Be Repeated Three Times (100 Per Well μ L Of HUVEC Cell Suspension). Coat 96 Well Plates With Growth Factor Reduced Matrigel. Melt Appropriate Volume Of Growth Factor Reduced Matrigel At 4 ℃ On The Day Before Use. Precool 96 Well Plates And Pipette Suction Heads At - 20 ° C For 2-3 Hours. Close To The End Of Serum Starvation Of HUVEC Cells. Before Counting HUVEC Cells, Divide Each Hole Of The Precooled 96 Well Plates Into 50 Equal Parts On Ice μ L Matrix Of Growth Factor Reduction. Rotate The Plate Until The Gel Is Evenly Distributed Over The Entire Hole. Avoid Bubble Formation. Allow It To Polymerize On The Horizontal Surface At Room Temperature For 1 Hour, And Distribute HUVEC Cells To The Coated 96 Well Plate For 100 μ L HUVEC Cell Suspension Obtained In Step 6 Of Procedure B Is Fully Mixed Into The Labeled Hole Of 96 Well Plate. Incubate The Plate At 37 ℃ And 5% CO 2 For 4-6 Hours To Visualize The Cells With A Light Microscope. Take An Image Of The Capillary Network And Use Scion Image Software To Calculate The Tube Length.