MUST

Cell counting and MTT assay

Hemacytometer
Introduction

A hemacytometer (also spelled hemocytometer) is an etched glass chamber with raised sides that
will hold a quartz coverslip exactly 0.1 mm above the chamber floor. The counting chamber is
etched in a total surface area of 9 mm2 (see Figure 1).

Calculation of concentration is based on the volume underneath the cover slip. One large square
(see W in Figure 2) has a volume of 0.0001 ml (length x width x height; i.e., 0.1 cm x 0.1 cm x
0.01 cm).

Method

To prepare the hemacytometer, the mirror-like polished surface is carefully cleaned with lens
paper and ethanol. The cover slips (which are thicker than those for conventional microscopy)
should also be cleaned. The cover slip is placed over the counting surface prior to adding the
cell suspension. The cell suspension is introduced into one of the V-shaped wells using a Pasteur
pipit. Allow the area under the cover slip to fill by capillary action. Enough liquid should be
introduced so that the mirrored surface is just covered. The hemacytometer is then placed on the
microscope stage and Count the number of cells in the 4 outer squares (see the left panel of
Figure 2).

Cell count formula:

Cell concentration per milliliter = Total cell count in 4 squares (W)/4 x 10000

Figure 1. Dimensions of a hemacytometer Figure 2. Counting procedure for Method

MTT assay
(Can be used for both Cell cytotoxicity & Cell proliferation experiment)

Introduction

The MTT assay is a colorimetric assay that relies on the enzymatic reduction of a yellow tetrazolium salt, 3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), which forms a purple formazan crystal in
metabolically active cells (Figure 1). The formazan can then be solubilized producing a concentration dependent
colorimetric signal at 570 nm proportional to the cell number and activity. This results in a sensitive assay that is
readily adaptable to a 96-well microplate format to analyze a large numbers of samples using a microplate
absorbance reader.

Cell suspension & dilution after Trypsin treatment

Cell suspension in medium, cell seeding in well plate & 24hrs incubation

Plasma treatment & incubation according to experiment requirement

Discrd media, PBS washing, and adition of MTT reagent as given bellow (light
protection)

Well plate (wp) MTT reagent Medium

6 wp 400 µl 1600 µl
12 wp 200 µl 800 µl
24 wp 100 µl 400 µl
48 wp 50 µl 200 µl
96 wp 25 µl 100µl

Gentle shaking, 4hrs incubation at 37C in dark (light protection)

Discard MTT & media, wash with PBS

 Add glycine and DMSO as given bellow (light protection)

Well plate (wp) Glycine buffre DMSO

6 wp 200 µl 1600 µl
12 wp 100 µl 800 µl
24 wp 50 µl 400 µl
48 wp 25 µl 200 µl
96 wp 12.5 µl 100 µl

Shake for colourful precipitate, load on 96wp (dilute with PBS if required)

Use DMSO as blank and take ELISA reading at 570nm (light protection)

Fig: MTT assay: ELISA reading

Crystal violet staining

(Can be used for both Cell cytotoxicity & Cell proliferation experiment)

Cell suspension & dilution after Trypsin treatment

Cell suspension in medium, cell seeding in well plate & 24hrs incubation

Plasma treatment & incubation according to experiment requirement

Stain with CV and incubate for 30 min at room temperature

Discard CV, and wash with PBS (2-3 times)

Add SDS and shake for colourful precipitate, load on 96wp (dilute with PBS if required)

ELISA reading at 610nm

Chemical preparation
A-Saline Solution preparation

Saline solution 0.085%: Mix 7.65g Nacl in 900ml distle water.

Autoclave for 25min at 121C.

B-Media

MEM 10% FBS and 1% antibiotics

C-Trizolyl blue tetrazolium bromide (TBTB)

50mg of (TBTB) per 10ml 0f 0.9% NaCl

D-Sorensen s glycine buffer preparation

0.1M glycine, 0.1 M Nacl adjusted to H 10.5 with NaOH

Crystal violet: 0.2% CV in 2% EtOH

Sodiumdodecyl sulfate: 0.5% SDS in 50% EtOH