MTT ASSAY Induction of Cell Proliferation by ConA

conA( concavalinA)  Lectin protein  Jack bean Canvalia ensiformis  lymphocyte mitogen LPS  Lipopolysaccharise pf Gram negative bacteria  Toll like receptor agonist

Mitochondria dehydrogenase enzyme MTT( yellow) dark blue formazan crystals (impermeable to cell membranes) NADH NAD+

Changes in NIH 3T3 cells morphology after exposure to MTT

Formazon crystal

water-soluble formazan MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) MTS phenazine methosulfate (PMS), water-soluble formazan

Materials: Raw 264.7( murine macrophage) Trypsin, culture medium MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] : Dissolved in RPMI-1640 at 5 mg/ mL and filter through 0.22 μm filters. Con A 4 ug/ml Sorensen’s Glycine buffer( 0.1M Glycine, 0.1M NaCl adjusted to pH10.5 woth 1M NaOH Acid isopropanol (0.1 N HCl in anhydrous isopropanol )

Calculation for cell plating: Plate map con conA 12 hrs 24 hrs Calculation for cell plating: Take 4x106 cells total, bring up volume to 2ml growth medium Add 200 ul cell /well( make 2x105 cells/well) Add conA( 50ug/ml stock) 20ul/well----- final 5 ug/ml

1. Plate 200ul of each tube on round bottom 96- well dish according to the plate map , incubate 37oC, 5% CO2 2 .Incubate 37oC, 5% CO2, till day 2( 48 hrs after) 3. After 48 hrs, add 15 ul of MTT to all wells of 48 hrs cultured splenocyte and incubate for 4 hrs at 37oC. 4. Pipette out the spent media along with suspension of cultured cells. 5. Then add 100 μL of acid isopropanol to all wells and mix thoroughly to dissolve the dark blue crystals 6. Incubate a 5 minutes at room temperature, 7. Read plates using a plate analyzer in dual wavelength test wavelength =540 nm, reference wavelength = 630 nm. Read plates normally within 1 h of adding the acid isopropanol. Calculate cell proliferation as stimulation index:                                         A540 nm with LPS      Stimulation index =                                          A540 nm without LPS

MTT assay for cell proliferation (MTT stock solution: 5mg/ml MTT) 1. Wash cultured cells with warm RPMI-1640 without phenol red. 2. Prepare MTT working solution. 3. Add MTT working solution into wells being assayed, for example 1.0ml for each well of 12-well plate. Incubate at 37°C for 30min to 3 hrs (this time depends on cell density and cell type). 4. At the end of the incubation period, the medium can be moved if working with attached cells. 5. The converted dye is solubilized with 1ml acidic isopropanol (0.04 M HCl in absolute isopropanol). Pipette up and down several times to make sure the converted dye dissolves completely.

6. Transfer the dye solution with the cells into a 1.5 ml eppendorf tube and centrifuge at 13,000 rpm for 2 min. 7. Transfer the supernatant into a new eppendorf tube. Absorbance of the converted dye is measured at a wavelength of 570nm with background subtraction at 650nm. For the

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Proliferation Assay Plate 2x105 cell/200ul/well Take 2x10 6 cells, bring up cells with 2 ml RPMI-1640( with glutamine)+ 5% FBS+ 50uM -Mecaptoethanol+ Penicillium/streptomycin Calculation for cell plating: Take 4x106 cells total, bring up volume to 2ml RPMI 1640( with glutamine)+ 5% FBS+ 50uM -Mecaptoethanol+P/S Add 200 ul cell /well( make 2x105 cells/well) Add conA( 100ug/ml stock) ----- final 5 ug/ml LPS ----- final 10 ug/ml