For my major project, I am required to culture and subculture mammalian cells. The type of cell I am culturing is large lung cancer cells; NCI-H460. This cell line grows best in RPMI medium.
Culturing of NCI-H460 cell line
1. Cryovial containing 1ml of NCI-H460 cells are removed from the liquid nitrogen and thawed in the 37°C water bath.
(Ensure that the cryovial is securely capped to prevent any leakage.)
2. The RPMI medium is warmed at 37°C water bath for approximately 30 minutes.
3. 5ml of the RPMI medium is added into the centrifuge tube.
4. All cells are removed from the cryovial and added into the 5ml RPMI medium.
5. The centrifuge tube is centrifuged at 1000rpm for 5 minutes.
6. The supernatant is removed and discarded into sodium hypochlorite.
7. The cell pellet is suspended in 10ml of RPMI medium.
8. 50µl of the cell suspension is removed and placed into a microcentrifuge tube (for cell counting).
9. The remaining cell suspension is placed into a T-75 flask.
10. 10ml of RPMI medium is added into the T-75 flask.
11. Incubate at 37°C with 5% CO₂.
Cell counting to determine seeding density
1. 50µl of the cell suspension is added into 50µl of trypan blue.
(Trypan blue will stain the dead cells)
2. Mix well by pipetting it up and down slowly.
(Ensure that the tip of the pipette is not placed against the surface to prevent damaging the cells.)
3. 10µl of the mixture is pipetted into the haemocytometer chamber.
4. Viable cells within the four corner squares of the haemocytometer are counted.
5. The cell density is calculated, average number of cells X 2 X 10
6. The total number of viable cells is calculated, cell density X 10ml
7. Clean the haemocytometer with alcohol.
(Ensure that it is not left for too long as the trypan blue will stain the haemocytometer.)
Strict aseptic techniques need to be observed as presence of contamination will prevent the growth of mammalian cells.
Liyana
0703827F
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Dear _____ (You are?),
ReplyDeleteHow many cells did you culture into the T75 flask?
And how long will the cell line take to be confluent?
Li Yinliang Alex
TG02 0704894E Group 8
22 October 2009
Hi
ReplyDeleteThe start seeding was 2.25 X 10^6 cells.
During the first few passage, it will take 3-4 days for the cells to reach 80-90% confluence. Gradually, as the cells adapt to the environment, it will take as fast as a day to reach 100% confluence.
Liyana
0703827F
hi liyana, just a bit curious here, how does trypan blue stain the dead cells? What is the principle behind it?
ReplyDeletezi shuang
Hi Liyana,
ReplyDeleteWhy is it that the cells grow best in the RPMI medium? What is the component of the medium that makes it the best?
Yvonee
0703189A
Hi zi shuang,
ReplyDeleteDead cells will lose their membrane permeability. Thus allowing trypan blue to enter the cell and stain it blue.
Liyana
0703827F
Hi Yvonee,
ReplyDeleteAccording to the ATCC RPMI media formulations, it constitutes of inorganic salts, amino acids and vitamins that are typical of other medias. But the type of constituents used differs from other media.
Example,
inorganic salts:
Ca(NO3)2·4H2O
KCl
Na2HPO4
vitamins:
Choline Chloride
myo-Inositol
Nicotinamide
amino acids:
L-Arginine
L-Asparagine·H2O
L-Aspartic Acid
It is also supplemented with HEPES, L-glutamine, sodium pyruvate and sodium bicarbonate.
RPMI media is best for this cell line, NCI-H460, due to the above components. All the components contributes to the growth of this cell line.
Liyana
0703827F