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

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 )

Plate map

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

con con con conA conA conA 12 hrs

24 hrscon con con conA conA conA

1. Plate 200ul of each tube on round bottom 96- well dish according to the plate map , incubate 37oC, 5% CO22 .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 crystals6. 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

http://www.youtube.com/watch?v=vn6enA6lSKs&feature=BFa&list=PLB7ECCD20439FEFFC&lf=results_main

MTT assay

http://www.youtube.com/watch?v=KoYpGaNvwi8&feature=related

http://www.youtube.com/watch?v=GU9byOFn4d8

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