MTT Practical Operation - Technical Experience

MTT Experiment Operation The Routine MTT Experiment Is Generally Drug Sensitivity Experiment And Cell Proliferation Experiment. The Success Of The Experiment Is Generally Related To The Uniformity, Consistency And Cell Activity Of The Cell Board& Nbsp; Preparation Method Of MTT Solution: 1 Preparation Method Of MTT Solution: Generally, The MTT Concentration In This Method Is 5mg/ml. Therefore, 0.5g MTT Can Be Weighed And Dissolved In 100ml Phosphate Buffer Solution (PBS) Or Medium Without Phenol Red. 0.22 μ M Filter Membrane To Remove The Bacteria In The Solution, And Store At 4 ℃ Away From Light. In The Process Of Preparation And Storage, It Is Better To Wrap The Container With Aluminum Foil. 2. Dissolve MTT With PBS (pH=7.4). PBS Formula: NaCl 8g, KCl 0.2g, Na2HPO4  1.44g，KH2PO4  Adjust The PH Of 0.24g To 7.4, And The Constant Volume Is 1L& Nbsp; Experiment Steps Of Drug MTT Method: Adherent Cells: 1 Collect Cells In Logarithmic Phase, Adjust The Concentration Of Cell Suspension, Add 100ul To Each Hole, And Make The Density Of Cells To Be Measured 1000 - 10000/hole By Planking. The Gun Head Of Cells To Be Added Each Time Sticks To The Periphery Of 96 Holes, And Slowly Add Them By Rotating. After Adding Cells, You Can Shake Them Horizontally From Front To Back, Left To Right For Several Times To Make The Planking Better. 2. Incubate With 5% CO2 At 37 ℃ Until The Cell Monolayer Is Covered With The Bottom Of The Hole (96 Hole Flat Plate). Add The Drug With Concentration Gradient. In Principle, The Drug Can Be Added After The Cell Adheres To The Wall. Generally, There Are 5-7 Gradients, 100 Ul Per Hole, 12 Holes In A Row, Two PBS Filled Holes, Two Control Holes, Two Blank Holes, And Six Multiple Holes. 3 Incubate With 5% CO2 At 37 ℃ For 16-48 Hours And Observe Under Inverted Microscope. 4. Add 10ul MTT Solution (5mg/ml, I.e. 0.5% MTT) Into Each Well And Continue To Culture For 4h. If The Drug Can React With MTT, The Culture Solution Can Be Discarded After Centrifugation. Carefully Rinse With PBS For 2-3 Times Before Adding The Culture Solution Containing MTT. 5. Stop The Culture And Carefully Suck Out The Culture Medium In The Hole. 6. Add 100ul Dimethyl Sulfoxide Into Each Hole, Place It On The Shaker And Shake It At Low Speed For 10min To Fully Dissolve The Crystals. The Absorbance Value Of Each Hole Was Measured At OD490nm Of The ELISA. 7. At The Same Time, Zero Adjustment Holes (culture Medium, MTT, Dimethyl Sulfoxide) And Control Holes (cells, Drug Dissolution Medium Of The Same Concentration, Culture Medium, MTT, Dimethyl Sulfoxide) Are Set. Suspended Cells: 1. Collect Cells In Logarithmic Phase And Adjust The Concentration Of Cell Suspension 1 × 106/ml (see The Following Precautions For The Cell Concentration), And In Order ① Replenish The 1640 (serum Free) Medium 40 Ul; ② Add 10 Ul Of Actinomycin D (toxicity) And Dilute It With Culture Solution (100mg/ml Of Storage Solution, Pre Test Is Required To Find The Best Dilution, 1:10-1:20); ③ 10 Ul Of Substances To Be Tested; ④ Cell Suspension 50ul (i.e. 5 × 104 Cells/hole), A Total Of 100ul Is Added To The 96 Hole Plate (the Edge Hole Is Filled With Sterile Water). Set Control For Each Board (add 100ml 1640) 2 Incubate At 37 ℃ With 5% CO2 For 16-48 Hours, And Observe Under Inverted Microscope. 3. Add 10 Ul MTT Solution (5 Mg/ml, I.e. 0.5% MTT) Into Each Well And Continue To Culture For 4 H. (WST-1 Is Recommended For Suspension Cells, And Step 4 Can Be Skipped After 4 Hours Of Culture). The Absorbance Value Of Each Hole Is Measured By Direct ELISA At OD570nm (630nm Calibration) Centrifuge (1000 Revolutions X 10min), Carefully Suck Out The Supernatant, Add 100 Ul Dimethyl Sulfoxide Into Each Hole, Place It On A Shaker And Shake It At Low Speed For 10min To Fully Dissolve The Crystals. Measure The Absorbance Value Of Each Hole At OD570nm (630nm Calibration) Of The ELISA& Nbsp; 5. At The Same Time, Set Up Zero Adjustment Holes (culture Medium, MTT, Dimethyl Sulfoxide) And Control Holes (cells, Drug Dissolution Medium Of The Same Concentration, Culture Medium, MTT, Dimethyl Sulfoxide), And Set Up 3 Double Holes For Each Group. Calculation Of Experimental Results Of Cell Proliferation: Cell Survival Rate%=(OD Of Drug Added Cells/OD Of Control Cells) X 100 The Spring Festival Is Coming. I Wish You More Successful Experiments And More Project Funds!