7. A post-doctoral fellow asks you to prepare 500 ml of RPMI media to culture RA

Question

7. A post-doctoral fellow asks you to prepare 500 ml of RPMI media to culture RAW cells. How do you prepare complete media containing 5% FBS and antibiotics? 264.7 The same post-doctoral fellow asks you to prepare a 6 well plate containing 10 RAVw 264.7 cells/well in OptiMEM media, Which additional reag ml OptiMEM media for it to be complete? What is the purpose of using OptiMEM? Describe a scenario in which it is useful to study cells in Go phase 8. ents need to be added to 500 You are asked to transfect RAW 264.7 cells with an siRNA targeting IL-12, a cytokine upregulated in cancer cells. The experiment is performed successfully, but as you are presenting the data, you discover the passage was at 30. Is the data reliable? Why or why not? 9.

Explanation / Answer

7. RPMI media with 5% FBS and antibiotics

For eg. if we need to make 100ml of RPMI media with 5% FBS and antibiotics.

5% FBS means 5 ml FBS for 100 ml

so take 5 ml FBS and add 95 ml RPMI media. It will now require only antibiotic to be added. Normally Penstrip antibiotic which is a Penicillin Streptomycin solution is added to the medium according to desired concentraiton,

8. To make the media complete we need antibiotic in addition to cells.Opti-MEM media is special type of medium which is used in the conditions, where we need to have presence of low serum in the medium. Opti-MEM is very useful for cell transfection experiments. During the cell cycle analysis it is useful to study cells in Go phase.

9. Passage no in cells means how many times the cell has been subcultured from the starting. So, passage no 30 means the cell line has beed subcultures 30 times before doing the experiments, Now, any cell culture, generally contained heterogenous cells. When cells are passaged several times, the genotype of cells can be changed from the original strain. It also can give rise to mix population of cells having different genotype than the referene strain. So it is always recomended to use low passage number cells for experiments like transfection. In this case, the data might not be as reliable as it was if done in a low passage number.

1. cell concentration =1.23 X 106 /ml.

required cell =100,000 cell / per well or 1 X 105 cells / well.

stock concentraiton=1.23 X 106 /ml or 12.3 X 105 cell /ml.

now,

12.3 X 105 cell are present in 1 ml

so 1 X 105 cells will be present in 1 X 105/12.3 X 105 = 0.081 ml or round about 80l

so when 80l from the stock solution will added to each well, it will contain 100,000 cell / well.

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