Cell Proliferation Assay

 

Introduction

Living (metabolically active) cells reduce tetrazolium salts to colored formazan compounds; dead cells do not. Thus, tetrazolium salt-based colorimetric assays detect viable cells exclusively. Because they are sensitive, these assays can readily be performed in a microplate with relatively few cells. Since a cytotoxic factor will reduce the rate of tetrazolium salt cleavage by a population of cells, these metabolic activity assays are frequently used to measure factor-induced cytotoxicity or cell necrosis. Applications include:

1. Assessment of growth-inhibitory or cytotoxic effects of physiological mediators
2. Analysis of the cytotoxic and cytostatic effects of potential anti-cancer and other drugs
3. Analysis of cytopathic effects of viruses and screening of compounds with potential
   anti-viral activity.
4. Screening of antibodies for growth-inhibiting potential.

Principle

Measurement of cell viability and proliferation forms the basis for numerous in vitro assays of a cell population’s response to external factors. The reduction of tetrazolium salts is now widely accepted as a reliable way to examine cell proliferation. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, first described by Mosmann in 1983, is based on the ability of a mitochondrial dehydrogenase enzyme (by generating reducing equivalents such as NADH and NADPH) from viable cells to cleave the tetrazolium rings of the pale yellow MTT and form a dark blue formazan crystals, which is largely impermeable to cell membranes, thus resulting in its accumulation within healthy cells. The resulting intracellular purple formazan can be solubilized and quantified by spectrophotometric means. Solubilisation of the cells by the addition of a detergent results in the liberation of the crystals which are solubilized. The color can then be quantified using a simple colorimetric assay. The results can be read on a multiwell scanning spectrophotometer (ELISA reader). The MTT Cell Proliferation Assay measures the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability. Here we took splenocytes for cell proliferation assay.
 

Reagents

Preparation of basal culture medium (RPMI-1640)

The following ingredients were added to one litre of autoclaved and cooled triple distilled water:
        Ingredients                 Amount
        RPMI 1640                 16.4 g
        NaHCO3                     2.10 g
        Sodium pyruvate         110.1 mg
        HEPES                        5.96 g (50 mM)
        L-Glutamine                293 mg
        Pencillin                      61 mg (100 IU/ mL)
        Streptomycin               100 mg (100 μg/ mL)

Adjust the pH of the resulting solution to 7.2 with the help of 1N NaOH and sterilize through 0.22 μm Millex-GV filter unit (Millipore). Store the prepared medium in small aliquots at 5° C in dark.
 

Erythrocyte lysis buffer

        10 ml of 0.17M Tris HCl
        90 mL of 0.6M of NH4Cl
        pH-7.2
 

MTT Solution

MTT was dissolved in RPMI-1640 at 5 mg/ mL and filter through 0.22 μm filters.

Procedure

Preparation of culture medium

Supplement the basal culture medium with 10% fetal calf serum (FCS-heat inactivated at 560 C for 30 min) for its use as culture medium and add mitogen i.e., Lipopolysaccharide (LPS; 10-50 μg/ mL) to proliferate the cells.
 

Preparation of splenocytes

1. Collect spleen tissues from mice aseptically and subsequently transfer to another sterile petridish containing medium.
2. Tease spleen tissues gently using a sterile needle and forceps to release cells from the tissues.
3. Allow it to stand for 2 min for sedimenting clumps.
4. Then transfer the upper portion of the medium containing the splenocytes using 1 ml pipette, to a 15 mL sterile centrifuge tube.
5. Again, leave to stand for 2 min (in ice) and transfer to a fresh centrifuge tube.
6. Centrifuge the cell suspension at 1000 x g for 5 min, wash cells once again in basal culture medium and then 1ml of erythrocyte lysis buffer was added to the pellet.
7. After 30 to 45 sec, add 5 mL of culture medium to this and centrifuge at 1000 g for 5 min.
8. Wash the lymphocytes twice with culture medium to remove the traces of lysis buffer and finally suspend in 1 mL of RPMI 1640 containing 10% FCS.
9. Count cells by using a small aliquot of cell suspension.

Dispensing and culturing of lymphocytes:

1. Make cell suspension containing 1x107 viable cells/ mL in culture media containing 10% FCS.
2. Dispense cells at the rate of 100 μL per well in 96 well flat bottomed tissue culture plates.
3. Allow cells to grow with and without mitogen (LPS; 10 μl/ well) to determine the difference between cell proliferation.
4. Incubate plates in a humidified CO2 incubator air atomphere (5:95) at 37° C for 48h.

Cell proliferation assay

1. Add 10 μL of MTT solution to all wells of 48 h cultured lymphocytes and incubate for 4 h at 37° C.
2. During this period, formazon crystals will be formed at the bottom of each well.
3. Pipette out the spent media along with suspension of cultured cells.
4. Then add 100 μL of acid isopropanol (0.1 N HCl in anhydrous isopropanol ) to all wells and mix thoroughly to dissolve the dark blue crystals.
5. After a few minutes at room temperature read plates using a plate analyzer in dual wavelength measuring system, at a test wavelength of 540 nm, a reference wavelength of 630 nm.
6. Read plates normally within 1 h of adding the acid isopropanol.
7. Calculate cell proliferation as stimulation index:

                                        A540 nm with LPS
     Stimulation index = -------------------------------
                                        A540 nm without LPS
          
            Where A 540 = Absorbance at 540 nm.