Published by : PROFESSIONAL MEDICAL PUBLICATIONS
April - June 2009 (Part-II)
Effect of Fetal Calf Serum on Cellular Proliferation of
Mouse Y1 Adrenocortical Cells in vitro
Muhammad Imran Naseer1, Hassan Zubair2, Ikramullah3, Myeong ok Kim4
Objective: Fetal calf serum (FCS) is a supplement used in cell culture media for successful culturing. The present study was design to observe the effect of FCS on cellular proliferation.
Methodology: Effect of different concentrations (0, 1, 5, 10 and 20%v/v) of FCS on cellular proliferation was determined by the uptake of crystal violet, MTT assay. Total cellular protein was also measured colorimetrically to observe the effect of FCS on growth of mouse Y1 adrenocortical cells.
Results: The results showed a gradual increase in proliferation of Y1 cells by increasing concentrations of FCS in Dulbecco’s modification of Eagle’s medium (DMEM). Highest proliferation rate of Y1 adrenocortical cells was achieved with cultured cells after 6 days in DMEM medium containing 10 to 20% FCS.
Conclusion: The study suggested that supplementation of 20% FCS increase cell proliferation and acts as a growth factor results in cell division and DNA synthesis.
KEYWORDS: FCS, cell proliferation. Y1 adrenocortical cells.
Pak J Med Sci April - June 2009 (Part-II) Vol. 25 No. 3 500-504
How to cite this article:
Naseer MI, Zubair H, Ikramullah, Kim MO. Effect of Fetal Calf Serum on Cellular Proliferation of Mouse Y1 Adrenocortical Cells in vitro. Pak J Med Sci 2009;25(3): 500-504.
1. Muhammad Imran Naseer, PhD
Gyeongsang National University,
Division of Life Science (BK 21),
Chinju 660-701, South Korea
2. Hassan Zubair, MS
Sheffield Hallam University,
Sheffield, S1 1WB, United Kingdom.
* Muhammad Imran Naseer & Hassan Zubair contributed equally to this work.
3. Ikramullah, PhD
Gyeongsang National University,
Division of Life Science (BK 21),
Chinju 660-701, South Korea.
4. Prof. Myeong Ok Kim, Ph.D
Division of Life Science, College of Natural Science
Gyeongsang National University 900,
Gazwa 351-301 Chinju 660-701, South Korea
Prof. Myeong Ok Kim, Ph.D
* Received for Publication: December 31, 2008
* Revision Received: June 4, 2009
* Revision Accepted: June 5, 2009
Fetal calf serum (FCS) is a supplement used in many cell culture media for successful culturing.1,2 It is known to contain growth factors and cytokines necessary for cell division.3 Supplementation of FCS in developing embryos results in DNA synthesis and cell proliferation.1,4-6
Growth factor signalling involves the use of multiple intermediate signals including calcium mobilization, inositol-phosphate formation and cAMP/cGMP formation. The initial signal sent by the ligand-receptor complex is converted into more widespread and general effectors, which are capable of initiating the multiple intracellular changes needed for cellular division.7-11 Addition of FCS to certain fibroblastic cells results in activation of DNA synthesis by increasing the concentration of intracellular Ca++. This observation clarifies the mechanism of growth activation.6
Addition of FCS to resting cultures cause immediate changes in transport rates and cyclic nucleotide concentrations, followed by enhancement of the rates of protein and RNA synthesis, and ultimately by a resumption of DNA replication. It has been suggested that the various changes in the response to serum are coordinate effects; however, since serum contains many different growth factors, and hormones, the various observable effects could be brought about by separate mechanisms.6
The aim of present study was to determine the effect of different concentrations of FCS at different time on proliferation of mouse Y1 adrenocortical cells.
Cell Culture: Y1 cells were grown in triplicate culture plates by using Dulbecco’s modification of Eagle’s medium (DMEM) containing 1-20% fetal calf serum, 2mM glutamine, 100µg/ml penicillin and 100 mg/ml streptomycin.6-16 Culture were maintained at 37C0 humidified atmospheres of 5% CO2 and 95% air.
FCS treatment on cell culture:The cells were removed from their substratum using trypsine (0.05%)-EDTA (0.02%) and were plated out at a density of about 0.25 x 105 cells/well (prepared in DMEM medium without FCS) into the center 60 wells of two 96 well plates, counted using a haemocytometer. Cells were allowed to settle overnight in the humidified gas incubator and then treated with different concentrations (0, 1, 5, 10 and 20% v/v) of FCS, 12 wells/treatment used for growth.
Crystal violet stain: Crystal violet method of measuring cell proliferation relies upon its uptake by the cells, which eventually stains DNA. The intensity of color produced is proportional to cell number. For the uptake of crystal violet, cells were allowed to proliferate in response to stimulatory agent for 48h. At the end of experiment, cell media were removed and cells were washed with 200 ml PBS. Cells were then fixed with 200 ml of methanol for 15 minutes. Crystal violet (0.1% solution in 200 mM boric acid) was added as 200 ml/well for 20 minutes and proliferation was determined by the intensity of color produced on solubilising the dye in 50 ml 10% glacial acetic acid for 30 minutes in gas incubator. Color produced was measured at 540nm using a plate reading multi-scan spectrophotometer.5
MTT Assay:The logarithmic growth phase of Y1 adrenocortical cells were taken for growth assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenly tetrazolium bromide (MTT). The MTT assay relies on the uptake of MTT, a water-soluble tetrazolium salt producing a yellowish solution. In cell cultures it is converted to an insoluble blue formazan by cleavage of the tetrazolium ring by dehydrogenases of mitochondria of living cells. The intensity of the blue color produced is a measure of viable cell number.5 For the MTT assay, cells were cultured with stimulatory agents for 48h and 20ml of MTT (5mg/ml solution in PBS) was added to each of the treated cultures 4h before the end of the experiment and cells were incubated at 370C in a humified 5% CO2 incubator. To achieve solubilization of formazan crystals formed in viable cells, DMSO was added to each well, and the absorbance was recorded on a micro titer plate reader at a test wavelength 570nm with a reference wavelength of 690 nm. The optical density (O.D) was calculated as a difference between the absorbance at the reference wavelength and that observed at the test wavelength. The effect of drugs on growth was assessed as percent of cell viability.17
Measurement of total cellular protein:Proliferation of Y1 adrenocortical cells was tested using a non-radioactive nucleotide analogue, 5-bromo-2'-deoxy-uridine (BrdU, Boehringer, Mannheim). Cells were seeded at a density of 10000/cm2 on flat-bottom 96 well micro titer plates in culture medium with or without 1 to 20% FCS, medium was changed every 48 hr. The cells were incubated for 16 hr with BrdU, rinsed twice with culture medium, fixed, washed and stained with an anti-BrdU antibody linked to peroxidase. The peroxidase cleavage substrate was detected colorimetrically at 490 nm versus 405 nm (dual wavelength ratio). The results of the BrdU measurements are expressed in arbitrary units (ratio x 1000).15
Total cellular protein was determined after 20 min of cell cleavage with Triton X100 1%v/v using a phenol reagent micro protein method (Sigma, Munich). Standards were prepared by dilution of bovine serum albumin. All data are expressed as µg protein per single well. The number of cells per well was determined by counting cell nuclei on photomicrographs of methylene blue stained cells on a standardized portion of the well bottom. Statistical significance was calculated using Student’s t-test, all data are given as arithmetic means±standard error.
Determination of cell proliferation by crystal violet stain: To asses the effect of FCS on cellular proliferation we studied the uptake of crystal violet by Y1 adrenocortical cells. The cells were grown as monolayer cultures and treated with different concentrations of FCS (0, 1, 5, 10 and 20% v/v) to stimulate cellular proliferation. The results showed that there was significant increase in proliferation of Y1 cells with the increase in amount of FCS in the DMEM medium. There was increase in absorbance value from 0.197 to 0.516 when concentration of FCS added in the medium increased from 0 to 20%.
To further study the maximum effect of FCS dose on cell proliferation we studied the crystal violet method. The results showed that a sharp increase in absorbance of Y1 cells was observed when 1% of the FCS was added in the DMEM medium, flowed by a gradual increase up to the addition of 10% FCS, finally a lower rate of increase in absorbance was achieved when concentration of FCS in the medium raised from 10-20% (Fig-1).
Determination of cell proliferation by MTT assay:
To further conform the cell proliferation effect by addition of FCS, we studied MTT assay. The results showed that there was a significant increase in absorbance value from 0.069 to 0.243 when concentration of FCS added in the medium from 0 to 20% . There was a gradual increase in absorbance value up to the addition of 5% FCS in the medium followed by a sharp increase in absorbance by the addition of 10 and 20% FCS (Fig-2).
Increase in total protein after FCS treatment
To asses the increase in total protein after 20% v/v concentrations of FCS on cellular proliferation, that may be the result of either cell enlargement (hypertrophy), or of cell proliferation, we counted the cells after application of FCS at 2 and 6 days after plating. On day 2, total cell number without FCS (3.10±0.15 x 104 cells/well) slightly differs from cells treated with 10% of FCS (4.70±0.15 x 104 cells/well). On day 6, FCS treated cells numbered 5.991±0.19 x 104 cells/well, an increase compared to the 4.10±0.10 x 104 cells/well of the controls, cells treated with 10% of FCS numbered 5.50±0.15 x 104 cells/well which was significantly different from controls. Total protein after 20% of FCS treatment rose from 90±6 to 150±4 pg/cell from the second to sixth day after plating (n=3) (Fig-3).
To further analyze the time and different concentrations of FCS treatments. The effect was detected from 2 to 6 days, but was most pronounced at an incubation period of 6 days. At this time, total protein doubled on 20% FCS (9.28±0.60 vs. 4.90±0.54 µg/well). Total 10 and 20% FCS treatment showed its maximum effect on total protein incorporation as compare to control group (Fig-3).
Proliferation capability and cell viability was quantitatively determined by the incorporation of crystal violet and MTT on living cells. In the present study, we observed that the crystal violet staining showed that adrenocortical cells actively proliferated with the addition of FCS under incubation of 1% of FCS resulting in a sharp increase in absorbance. Further, cells proliferation was confirmed by MTT assay, gradual increase in absorbance was found up to the addition of 5% FCS. This suggested that although proliferation rate was same as in crystal violet method, but there was less formation of blue formazan product due to the presence of less amount of mitochondrial dehydrogenase. However no reduction in increase in absorbance rate during 10 to 20% FCS addition observed in crystal violet and MTT assay (Fig-1 & 2).
An overall increase in absorbance by Y1 adrenocortical cells achieved due to the stimulation of cell proliferation by the addition of FCS in crystal violet, MTT assay and total cellular protein was also measured colorimetrically. Previously, Bertsch and Marks,4 Langendonckt et al.,9 and May et al.,10 reported increased cellular proliferation with the addition of FCS in culture medium. Increased proliferation rate of adrenocortical cells by the addition of FCS occurred due to multiple effects because of the presence of epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) in FCS.10,12,14 These multiple effects include calcium mobilization, inositol-phosphate formation and cAMP/cGMP formation due to growth factors signalling. The initial signal sent by the ligand-receptor complex is converted into more widespread and general effectors, which are capable of initiating the multiple intracellular changes needed for DNA replication and cellular division.7 Finally, the results indicated that FCS contained growth factors necessary for cell division and is a strong inducer of cell proliferation.
This work was supported by KOSEF, grant funded by the Korean government (2009-0058805) and Brain Korea 21.
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