Observation Report

Basic Steps Involved in the Preparation of a Cell-based Assay

By Swasti Chaudhary

Cell-based assays are used in biomedical research to quantify cytotoxicity, biological activity,
biochemical mechanisms etc. They are used to evaluate and assess the cell health in mammalian
cell lines, stem cells and primary cells.

Introduction
The tissue of origin is most accurately represented by primary cells. They are extracted straight from the
tissue and then prepared for optimal culture conditions. They demonstrate typical physiology and are more
akin to the in vivo condition because they are produced from tissue and not changed. They thus make good
model systems for research into the typical physiology and biochemistry of cells (such as studies into
metabolism, ageing, signaling, and the impact of medications and harmful substances on the cells).

Cell culture is discovering its use in molecular and cellular Biology. It provides scientists with the right tools
to study the physiology and biochemistry of various cells.

1: Revival
Before beginning, in order to aid the cell’s survival, it is
important to perform each step quickly and under the optimal
conditions of temperature and medium formulation. For
revival, the vial of cells is thawed in a water bath at 37
degrees Celsius. Using a pipette, we transfer the contents of
the vial to a centrifuge tube, further, we gradually add 10ml
of pre-warmed medium to the tube. We then spin the content
in a centrifuge to remove the freezing medium. Once again,
using a pipette we discard the medium and re-suspend the
cells in an appropriate volume of pre-warmed medium. Suspending the cells in an appropriate medium
after they have been spun in the centrifuge.
2: Maintenance
The maintenance phase begins only when cells get attached to the culture flask, this usually takes up to 24
hours after initiation of the culture. Here, it is important we remove the spent media because DMSO
(cryoprotectant) can be harmful to primary cells and cause a drop in viability after revival. When cells
reach the desired confluency (the percentage of the surface area that is covered with cells) it is time to sub-
culture the cells. This should ideally be done before reaching 100% confluence.

A variety of cells showing

different amounts of
confluency.
 3: Trypsinization and Harvesting
The materials needed for trypsinization are serological pipettes, pipettes, tubes, tube rack, pipette tips,
waste bottles. We must begin by examining the culture for signs of deterioration and contamination. Cells
should be 70-80% confluent and healthy for optimal results.Taking the culture flask from the incubator, we
completely discard the old media. To rinse the cells, we use PBS or Phosphate-buffered saline. This is
done to remove traces of the serum that would otherwise inhibit trypsin.

For cell detachment, we then add enough pre-warmed trypsin

to cover the surface of the flask, and swirl the flask to ensure
full coverage. Then, we incubate the cells for a few minutes
before examining them for rounding up and detachment. The
incubation period may vary depending on the cell line. After
adding the complete growth media over the cell layer, we
must collect all cells in suspension and centrifugate them.
Now, we remove the supernatant which leaves us with the
cell pellets only. We will then dilute the cells in new media
and place the newly diluted cells in new plates or flasks
allowing them to grow. While putting the cells in the centrifuge
we must ensure that they are balanced by
4: Counting and Plating using a dummy tube if necessary

Hemocytometers are commonly used to estimate cell number and determine cell viability with the aid of an
exclusion dye such as Trypan Blue or Erythrosin B. A hemocytometer is a fairly thick glass slide with two
counting chambers, one on each side. Each counting chamber has a mirrored surface with a 3 × 3 mm2 grid
consisting of 9 counting squares.

Guideline for counting cells using a hemocytometer (left) Hemocytometer (right)

We must use a cell scraper to scrape off any cells that may have settled in the flask while waiting for the
count once the cells have been resuspended in the appropriate amount of growth fluid. Then, we pour
some cell suspension into a petri dish that will be used for plating.

5: MTT Assay
MTT assay is an assay that provides a readout for cell growth
and viability that works by measuring cell metabolic activity.
MTT is a soluble, powdered reagent that is added to cells and
incubated. Any viable or proliferating cells contain NADPH
enzymes which naturally reduce MTT.

MTT Assay
When MTT is reduced, insoluble crystals called formazan are generated. These are purple crystals that
form at the bottom of the cell plate. After incubation, once the crystals have formed, we carefully remove
the media and dissolve the crystals in DMSO to solubilize them. We then obtain a purple solution.

This purple solution will change its colour and intensity based on how viable the cells are and how many
crystals are formed. If a few crystals are formed then that implies that the cell is mostly dead or not
growing whereas if the solution is deep purple and the cells are growing well we can say that the solution
as a 100% viability. This is how the MTT assay helps in the quantification of growth and viability.

Cell Lines Observed:

Cell Line Medium

HaCat
Dermal Cell Basal Medium
Human Keratinocyte

A549 F-12K Medium (Kaighn's Modification of

Lung Cancer Ham's F-12 Medium)

Eagle's Minimum Essential Medium

SH-SY5Y
(EMEM)
Neuroblastoma

MCF7 Eagle's Minimum Essential Medium

Breast Cancer (EMEM)

Acknowledgements
I would like to thank the following people for giving me the opportunity to observe the proceedings of
the lab, and for taking the time to explain the procedures and projects being worked upon. I am grateful
for this experience and for al that I have learnt over the course of my training at the Dabur Research
Foundation.

Dr. Anu T. Singh Dr Alka Madaan Neha Gupta

Chief Scientific Officer Head of Cell Biology Lab Senior Research Scientist
Dabur Research Foundation Dabur Research Foundation Dabur Research Foundation

Karnika Saurabh Khurana

Research Scientist -II Research Scientist -I
Dabur Research Foundation Dabur Research Foundation
 This is to certify that Swasti Chaudhary completed her training in Dabur
Research Foundation in Cell Biology Department

Certified By:

Dr. Alka Madaan

Head Cell Biology Lab
Dabur Research Foundation