Frozen cells or cryopreserving the cells

Cryopreservation

People doing cell cultures often end up having more cells than needed or might not need the cells at that point of time. In such cases, the cells can be stored by freezing for later use. The process of preserving the cells by freezing is known as cryopreservation. The freezing stocks can be stored in liquid Nitrogen for an indefinite period.

Freezing can however, cause damage to cells by the formation of ice crystals, pH change. To avoid this, a cryopreservative agent such as DMSO (Dimethyl sulfoxide) was added to cells before freezing. DMSO lowers the freezing point and prevent ice crystal formation inside the cells. 

The freezing process is carried in 2 steps

• The cells were cooled slowly from room temperature to -80 degrees C either in a freezing chamber or  Mr Frosty in -80 degrees C freezer . Mr.Frosty is just a container filled with isopropanol. The advantage of Mr.Frosty is controlling the rate of cooling that helps freezing the cells successfully.  The cooling rate in this container will be repeatable -1 degrees C/min. 
•  After leaving at -80 degrees C for a day, the vials with frozen cells are transferred to liquid nitrogen (-196 degree C). 

Difference between primary cultures and cell line

People without previous knowledge on cell cultures often might wonder what are primary cultures and cell lines.

Primary culture:

• These are the cells that are derived directly from the tissues (enzyme digestion) or from tissue fragments (also known as explants). 
• It is very hard and time consuming to grow and maintain primary cell cultures compared to cell lines. 
• As they retain the characteristics and reflect the true activity of the cell type in vivo, they are preferred for the cell culture experiments (For example; To understand the disease, primary cells are grown from both diseased patient biopsy (tissue affected by the disease - for example skin tissue) and healthy volunteers skin tissue and differences are studied by performing various assays or experiments. 
• The drawback of the primary cells is their life-span. They can only be grown for a limited period of time.
• The cells obtained will depend on the tissue that are used to grow them (For example when used endothelium of blood vessels, endothelial cells and when used connective tissue, fibroblasts are obtained). 
• Usually the cells grown from tissues at the starting stage will have a mixture of cells (heterogeneous population). The media is given in such a way that only the cells that you intend to grow survives and rest die.

Cell lines (continuous):

• These are the transformed cells that has the ability to grow continuously (infinitely).
• They are transformed or changed by many ways (treat with carcinogens, expose to virus).
• As they are transformed, they might loose some of their original in vivo characteristics and might not completely represent the cells of the tissue that they come originally come from.
• The advantages are they grow fast, continuously and need less serum in media. 
• As a result, many experiments can be run in short time without worrying about cells dying after certain time.

Example of cell lines:

HeLa -----Epithelial cells

THP-1 ----Monocytes

NIH/3T3--Fibroblasts

BAE-1 ----Endothelial cells

CHO ------Fibroblasts

Buffer solutions

Buffer solutions are routinely used in all labs. What are these and why are they used.

A buffer solution is the one that resists a change in pH on addition of either acid or base.

Why are they important: Biochemical work in the lab usually involves biochemical molecules which are weak electrolytes. Their ionic status varies with pH and so there is a need to stabilize the ionic status during the course of the experiment.

How it works: It consists of an aqueous mixture of a weak acid and its conjugate base. During the experiment, any hydrogen ions generated are neutralised by conjugate base component while any bases generated are neutralized by unionised acid.

For more information, watch this video from MIT

https://www.youtube.com/watch?v=HZFIdpThd-s

Ready made formula to calculate the weight of chemicals to prepare Molar solutions (beginners/starters)

It could be a bit tough for the beginners to make solutions involving Molar concentrations. People might get confused and internet search might confuse them further. Actually, it is very easy and straight forward. If you apply the below fomula (easy to remember), you get to calculate the amount of solutes (weight in grams).

A = VMG
      1000

A = The amount (weight) that you want to calculate to make up the Molar solution (in this formula, always in gm or grams)
V = The volume of the solution that you intend to prepare (in this formula, always in ml or milli litre)
M = The molarity (you would know it as 1M or 2M or 0.1M or anything that you are looking
G = Gram molecular weight of the compound (you can find it on the chemical bottle in your lab- it might say formula weight or molecular weight)

For example : Make or prepare 500ml of  0.1M NaCl

A = 500 x 0.1 x 58.44   = 2.922 gms
              1000
So you take 2.922gms of NaCl in 500ml of water to get 0.1M solution of sodium chloride

Role of SDS, β-mercaptoethanol, bromophenol blue and glycerol in western blot sample buffer or laemmli buffer

β-mercaptoethanol is a reducing agent - it reduces any disulphide bridges that are holding the protein tertiary structure.

SDS -Sodium dodecyl sulphate is an anionic detergent that strongly binds to proteins and denatures it. On average one SDS molecule binds for every 2-amino acid residues. SDS molecules bound along the polypeptide chain gives the proteins a net negative charge. The treatment opens up the proteins into a rod-shaped structure.

Bromophenol blue is an ionisable tracking dye. The dye helps in monitoring the electrophoretic run. Otherwise, the proteins that run cannot be tracked and consequently the proteins might run off into the buffer at the bottom.

Glycerol or sucrose are used to give the sample solution density (add weight to the sample). This allows the sample to settle easily when loaded into the well.

Hybridomas

Hybridomas are produced by the fusion of antibody secreting B cells with a cancer cell line. Hybridomas has immortality conferred by the cancer parent cell. As B cells have a limited life span in cell culture, hybridomas are very helpful to produce monoclonal antibodies.

Basic difference between Polyclonal and Monoclonal antibodies

Monoclonal Antibodies:

1. The antibody comes from a single cell clone (Monoclonal).
2. These are epitope specific i.e., they can bind to only one epitope on an antigen.
3. Staining by using them gives less background as they have only specific antibodies.
4. They are produced artificially by animal cells in tissue culture which are known as hybridomas.
5. The production is well controlled hence, the antibodies don't have batch to batch variability

Polyclonal Antibodies:

1. The antibody has been produced by many clones (Polyclonal).
2. These are not epitope-specific i..e., they can bind to multiple epitopes of an antigen.
3. Staining by using these antibodies might sometimes produce background signal due to presence of non-specific antibodies.
4. They are derived from animal blood serum which were injected with antigen. 
5. These antibodies might have batch-batch variability.

Due to several of above reasons, monoclonal antibodies are more expensive compared to polyclonal antibodies.

Important note: Epitopes (known as antigenic determitant)

Normally we learn that antibodies are produced against antigens but you need to understand that antigens have several epitopes and antibodies are produced against these epitopes of an antigen but not the whole antigen. As polyclonal antibodies are produced in animals by injecting antigen, antibodies are produced against several epitopes of injected antigen, hence when collected they have antibodies which are antigen- specific but not epitope-specific. The concept of monoclonal and polyclonal becomes clear when we understand this difference. To make more sense, please see the figure below

Difference between avidity and affinity of antibody

The terms 'avidity' and 'affinity' are used in the context of antigen-antibody interactions

The strength by which the antibody binds to the antigen - Avidity
The 'fit' of the antibody shape to the antigen - Affintiy