19th week of SIP- Lab Techniques( Research)

hi, time passes and we will be back to skool soon...
tis time, I am going to discuss about cell transfection and cell lysis.

Cell transfection:

It is the introduction of a foreign gene into mammalian cultured cells. My main purpose of the cell transfection is to obtain the maximum protein yield from the cells.

Applications of cell transfection: to make proteins for clinical or research applications
Changing the protein expression profile of a cell to assay for the effects of a gene to study for the cell physiology, endogeneous proteins, phenotypes or over expression of the proteins and addition of genetic markers to a cell line
The cell transfection is done by introducing a maximum amount of 3 ug of DNA but it also varies between cell lines. The cell line that I am using is the HeLa cell and they can become confluent between a day. Once they reach 20 – 30 millions cells, we must not allow them to grow anymore or they can mutate since the HeLa cells are immortal.

Cell transfection can be accomplished through several methods, including microinjection of foreign DNA into the cell or through a chemical or biological reagent. However, in my lab, we only introduce the DNA using the chemical reagent, lipofectamine and opti-mem.

Procedures:

Cell transfection can only take place in an Antibiotic- free media. A day before transfection, 1.2 millions cells are aliquoted into a 60 mm dish and resuspended in an antibiotic-free media. Transfection in a 100mm dish is time consuming and uses more reagents and DNA, thus only 60mm dish is used for transfection.

After 8 hrs from the time of transfection, the old antibiotic- free media is replaced with complete media.
After 24-48 hrs, the cells are lysed, harvested and quantitated with Bradford assay.

Things to note before and after cell transfection :
the cell density and its appearance: Cells must reach 70-90% confluency during transfection and during lysis. Anything that is not within the range will cause the cells to die due to toxicity. Besides, we need to monitor for cell numbers after 24-48 hrs from the time of transfection as there might be cell death and we must make sure that the cells that are still alive is enough for the next step- cell lysis. Cell death can occur often in cell transfection due to the stress they undergo. Cell abnormalities must also be observed.

Cell lysis

After 24-48 hrs of ill- treating the cells with the foreign DNA and reagents, the cell are ready to be lysed!!! Hahahaha… cell lysis is done with the lysis buffer with protease inhibitor (PI). There are different ways of cell lysis and will be mentioned later.

Procedures:

Method 1: cell scrapping

The old media is removed. The dishes will be rinsed with phosphate buffer saline (PBS). Lysis buffer is pipetted into the dishes and cells are scrapped with a cell scrapper. The contents left inside the dishes are transferred into a new tube and left to stand on ice for 10 mins.
NB: After harvesting the cells out from the dishes, all tubes must be kept chill at 4C.

After the 10 mins of incubation, the tubes are spun at 13000 rpm for 10 mins at 4C.
After spinning, the supernatant is pipetted out into a new tube and we can quantitate the protein concentration for our pull down assay and load for Western Blot analysis. Normally, lysate from cell scrapping may gives a false high protein estimation.

Method 2: passing thru the syringe

The old media is removed. The dishes are trypsinized twice with 1 ml of trypsin and harvested with PBS and the PBS- resuspended cells are transferred into new tube. The tubes are spun for 5 mins at 6000rpm and the supernatant is removed. The remaining pellet is dipped into liquid nitrogen and then, thawed in ice. After which, the pellet is resuspended with lysis buffer. An insulin syringe will be used to puncture the cells through the syringe to create pressure for lysing the cells. The tubes are allowed to stand in ice for 10 mins and centrifuged for another 10 mins. Supernatant is removed and transferred into new tube and ready for quantitation.

Method 3: sonication

Instead of using an insulin syringe to lyse the cells, sonication can also be used to lyse the cells. the tubes are allowed to stand in ice for 10 mins and sonicated. after which, the tubes will be spun for 10 mins to harvest the cells. However, the number of pulses and how long it takes for each pulse is an important factor affecting on protein yield.

Thats all for my last blog for SIP!! take care..n enjoy last few days of work!!

Ai Tee
TG 01
0503160D