Full Step Cell Invasion Experiment

1、 Material Preparation Photographic Microscope, Transwell Chamber, Aperture 8 μ M. Transwell Without Coating Glue, Cell Culture Plate For Migration Experiment 24 Well Plate. The Cell Culture Plate Shall Be Matched With The Purchased Transwell Cell, BD Company's Matrix, Serum Free Basic Medium, Normal Complete Culture Medium, Sterile PBS, Cotton Swab, Trypsin, 4% Paraformaldehyde Fixative Solution Or Methanol, Crystal Violet Dye Solution (0.1% PBS Crystal Violet) II. Steps And Processes 1. Dilute With BD Company's Matrix 1:8 Or According To The Amount Of Mmp Generated By Cells, And Coat The Upper Chamber Surface Of The Bottom Membrane Of The Transwell Cell, Set At 37 ℃ For 30min To Polymerize Matrigel Into Gel. Hydrate The Basement Membrane Before Use. 2. Before Preparing The Cell Suspension, The Cells Can Be Starved For 12-24 Hours With The Complete Culture Medium With Only 1% Serum Added To Further Remove The Influence Of Serum. 3. Digest The Cells, Centrifugate And Discard The Culture Medium After The Digestion Is Terminated (wash With PBS For 1-2 Times), Resuspension With Serum Free Medium Containing 0.1% BSA And Adjust The Density, And Generally Adjust The Cell Density To 5 × 10^5cells/ml。 4. Take Cell Suspension 100 μ L When Adding Transwell's Upper Chamber, It Can Also Be Adjusted According To The Cell Growth Rate. The Operation Tips: The Invasive Capacity Of Cells With Different Inoculation Doses Is Different. If There Are Too Many Cells, The Cells Passing Through The Membrane Will Be Too Many And Too Fast, And It Will Be Difficult To Count The Results Finally; The Number Of Cells Is Too Small, And It May Not Be The Time Point For Detection. All Cells Have Passed Through And Entered The Lower Chamber. Therefore, At Least A Certain Amount Of Cells Should Be Kept Indoors When Collecting Samples. 5.  24 The Lower Chamber Of Orifice Plate Is Generally Filled With 600 μ L Complete Culture Medium Containing Growth Factors Or Serum. Special Attention Should Be Paid To The Fact That There Are Often Bubbles Between The Lower Culture Medium And The Chamber. Once Bubbles Are Generated, The Chemotaxis Of The Lower Culture Medium Will Weaken Or Even Disappear. Once Bubbles Appear During The Seed Plate, The Chamber Should Be Lifted To Remove The Bubbles And Then Placed Into The Culture Plate. In Case Of Parallel Holes, Two Groups Are Generally Set. 6. Cultured Cells: Routinely Cultured For 12-48h (mainly Depending On The Invasiveness Of Cells). 24h Is More Common. In Addition To The Invasiveness Of Cells, The Influence Of Processing Factors And Cell Number Cannot Be Ignored When Setting Time Nodes. 3、 Result Statistics Steps: 1; Take Out The Transwell Chamber By Direct Counting Method, Discard The Culture Solution In The Hole, Wash It Twice With Calcium Free PBS, Fix It With Methanol For 30 Minutes, And Air Dry The Chamber Properly. 2. Dye With 0.1% Crystal Violet For 20 Min, Gently Wipe Off The Upper Layer Of Non Migration Cells With A Cotton Swab, And Be Careful Not To Rub The Layer Of Cells That Have Penetrated The Membrane, And Then Wash It With PBS For Three Times. 3. Take 6-10 Visual Fields Of Each Sample Under 400 Times Microscope To Observe The Cell Count, Take The Average Value, And Make Statistical Analysis