Measuring Cytotoxicity in HepG2 Cells

Measuring Cytotoxicity in HepG2 Cells Measuring cytotoxicity in HepG2 cells treated with cisplatin or 5-fluorouracil, via flow cytometry as a measure of effectiveness of the drugs at treating cancer Introduction Cytotoxicity is the quality of being toxic to cells, resulting in cell death (Rang et al. 2012). In the majority of therapeutics cytotoxicity is not a desirable characteristic. However, in the treatment of cancer many chemotherapy drugs, through cytotoxicity are designed to selectively induce apoptosis, a form of cell death (Rang et al. 2012). There are three main forms of cell death, these are: apoptosis, autophagy and necrosis. Apoptosis is the physiological process by which cells are destroyed in a controlled manner (Elmore 2007). There are four basic steps involved; the first is cell shrinkage, blebs begin to form and condensation of chromatin occurs. Next, membrane blebbing occurs and enzymes begin the process of nuclear fragmentation. In the late stages of apoptosis, the cell is broken down into apoptotic bodies which contain the cells components and nuclear fragments. The final stage is the phagocytosis of apoptotic bodies (Ulukaya et al. 2011). One of the main ways in which phagocytes recognise apoptotic cells is the presentation of phosphatidylserine (PS). Ordinarily, this phospholipid is found on the inner leaflet of the membrane bilayer, during apoptosis it translocates to the outer leaflet and binds to a PS receptor present on the phagocyte (Wu et al. 2006). In autophagy, targeted components such as organelles are isolated in an autophagosome; a form of vesicle with a lipid bilayer membrane. These autophagosomes are degraded by lysosomes (Kroemer, G et al. 2009). Necrosis is often thought of as uncontrolled cell death. However, there is an increasing amount of evidence that suggests it is regulated by transduction pathways and other mechanisms (Kroemer, G et al. 2009). It is caused by factors such as trauma, infection and toxins. It is characterised by the swelling of cells which causes the cell membrane to rupture, resulting in the release of intracellular components. These intracellular components can trigger a local inflammatory response. In contrast to apoptosis, the phagocytic stage does not occur in necrosis (Golstein, Pierre and Kroemer, Guido 2007). As a result, dead cells and their intracellular components remain at the site of necrosis, this build-up of debris may lead to conditions such as gangrene. Due to its detrimental effects, it’s preferential that necrosis is not produced by therapeutics. Cisplatin and 5-fluorouracil are chemotherapy agents, which both cause apoptosis by interfering with DNA via its replication and repair. Cisplatin crosslinks with purine bases in DNA which interferes with DNA repair mechanisms, this means that DNA damage is not fixed triggering apoptosis (Dasari and Bernard Tchounwou 2014). 5-flurouracil is an antimetabolite, it is metabolised into 5-fluorodeoxyuridine monophosphate (5-FdUMP) which is then incorporated into both DNA and RNA and also inhibits thymidilate synthase (Parker and Stivers 2011). HepG2 cells are derived from the hepatocellular carcinoma cells of a 15 year old, American, Caucasian, male and are epithelial in nature. They are commonly used as an in vitro model of human hepatocellular carcinoma (Costantini et al. 2013). Flow cytometry is a powerful tool that allows for the rapid differentiation and characterisation of particles including cells. A flow cytometer is made up of three main components: fluidics, optics and electronics. When a sample is injected into a flow cytometer, the fluidics system is responsible for producing a stream of single particles, this relies on the principle of laminar flow. This allows the particles to be interrogated by the laser beam individually. When a particle passes through the laser beam it scatters light. The amount of forward scatter is proportional to the size of the cell. The side scatter is proportional to the complexity of the cell. If a fluorophore is present, it can be excited by a specific wavelength of light. It will then undergo a Stokes shift and a different wavelength of light will be emitted which can be measured by the electronics of the flow cytometer (Macey 2007, Maxwell and Hannon-Fletcher 2009). The fluorophore can be conjugated with proteins suc h as annexin V which have a known high affinity to specific molecules present on the cell surface. Annexin V is a 35.7kDa, calcium sensitive, phospholipid binding protein. It has a high affinity to phosphatidylserine. The binding of fluorophore labelled annexin V is used as a common measure of apoptotic cells by flow cytometry (van Genderen et al. 2008). Aims To measure the effectiveness of two anti-cancer drugs by measuring the level of apoptosis in a population of HepG2 cells after being treated with cisplatin (15 µg/mL) or 5-fluorouracil (25 µg/mL) for 48 hours and compare those to a population of untreated cells via flow cytometry. Objectives To count and fix a population of HepG2 cells before staining them with fluorescently labelled Annexin V in preparation for analysis using a flow cytometer. To measure the level of fluorescence in 5000 treated and untreated cells using a flow cytometer as well as measuring the minimal and maximal fluorescence in a negative and positive control. Based on the fact the fluorescence is directly proportional to apoptosis determine which treatment is significantly better and provide evidence from the literature as to why this is the case. Methods Please refer to laboratory schedule (Hatch 2014). Following the collection of data descriptive statistics and a one-way ANOVA test were carried out the results of which can be found in the appendix. Results The negative control was used to identify if there was any background fluorescence. In table 1 (found in the appendix) the mean fluorescence for the negative control was 144. Therefore, fluorescence that is significantly higher than this level can be attributed to the excitation of the fluorophore (Alexa 488). Figure 1 Cytotoxicity of HepG2 cells measured as fluorescence using flow cytometry. The bars are means of triplicates, the standard error has been used for the error bars. *statistically significant compared to control (blue for negative, red for positive), p In figure 1, a greater level of fluorescence was seen for the samples treated with cisplatin and 5-fluorouracil compared to that of the untreated sample. Cisplatin produced the greatest level of fluorescence of the two treated samples. Discussion The data used was a class set, some of the data provided was produced by those running the laboratory session as some samples did not contain a pellet which was needed to run the flow cytometry analysis. The five sets of data were compared with one another, it was found that the differences were statistically significant (p As mentioned in results, cisplatin showed a greater mean fluorescence compared to 5-fluorouracil, Results were 726 and 540 respectively. Seeing as fluorescence is directly proportional to phosphotidylserine expression, which in turn is a direct measure of apoptosis. It can be assumed that cisplatin produced the highest level of apoptosis and is most cytotoxic to HepG2 cells. Although a higher concentration of the drugs were used (150 µg/mL) this result has also been seen in the literature (Brenes et al. 2007). Although it was not stained with annexin V, the negative control showed a mean fluorescence of 144; this is due to auto fluorescence of the cells. The fluorophore which was conjugated with annexin V was Alexa 488. It fluoresces at a wavelength of 488nm, this is within the green part of the spectrum. It is well documented that molecules found within all cells, including hepatocytes also fluoresce at a green wavelength, the most notable of these are flavins which fluoresce at around 560nm (Croce, Anna Cleta et al. 2007, Croce, Anna C et al. 2014). FAD (flavin adenine dinucleotide) is an example of a flavin and is a redox cofactor used in metabolism. HepG2 cells are a liver carcinoma cell line. Due to their cancerous nature there would be a high level of metabolism within these cells and hence, a high level of FAD. The low level of fluorescence seen in the untreated sample is expected as there should be no apoptosis induced by cytotoxicity. Therefore, it is not surprising that the difference between the fluorescence of the untreated sample and the negative control and the treated samples is less statistically significant. In this experiment, only one stain was used in this experiment (Annexin V), this allows for the identification of cells that are cytotoxic. To evaluate the true effectiveness of a chemotherapy drug, the cell viability and necrosis is also needed. Necrosis and late stage apoptosis can be measured using propidium iodide (PI). PI binds to nucleic acids and is impermeable to intact membranes, it is often used in combination with annexin V as it fluoresces red and there is little overlap between the wavelengths of red and green light. Therefore, compensation is not needed after flow cytometry has been carried out. It has been observed in the literature that the cytotoxicity of 5-fluorouracil is dependent on both dose and exposure time (Okamura et al. 2008). We only looked at one dose and exposure time for both drugs. It has also been observed that the use of cisplatin in combination with 5-fluorouracil or other chemotherapy drugs has a synergistic and additive effect in hepatoma cell lines (Kogure et al. 2004), both of these could be investigated further is the experiment were carried out again. Conclusion In conclusion, cisplatin was shown to produce a higher level of apoptosis in HepG2 cells than 5-fluorouracil. However, to fully evaluate the effectiveness of an anti-cancer agent, measures of viability and necrosis are also needed. The differences between the two drugs may be due to the different mechanisms by which the drugs work. The introduction of 5-dUMP into DNA and RNA is a more subtle mechanism compared to the cross-linking of DNA via cis-platin. This cross-linking may be detected by cells more readily resulting in higher levels of apoptosis. As HepG2 cells were used, these results may only be the case for hepatocellular carcinomas. References Brenes, O., Arce, F., Gà ¤tjens-Boniche, O., and Dà ­az, C. (2007) â€Å"Characterization of Cell Death Events Induced by Anti-Neoplastic Drugs Cisplatin, Paclitaxel and 5-Fluorouracil on Human Hepatoma Cell Lines: Possible Mechanisms of Cell Resistance.† Biomedicine pharmacotherapy = Biomà ©decine pharmacothà ©rapie 61 (6), 347–55 Costantini, S., Di Bernardo, G., Cammarota, M., Castello, G., and Colonna, G. (2013) â€Å"Gene Expression Signature of Human HepG2 Cell Line.† Gene 518 (2), 335–45 Croce, A.C., Ferrigno, A., Santin, G., Piccolini, V.M., Bottiroli, G., and Vairetti, M. (2014) â€Å"Autofluorescence of Liver Tissue and Bile: Organ Functionality Monitoring during Ischemia and Reoxygenation.† Lasers in surgery and medicine 46 (5), 412–21 Croce, A.C., De Simone, U., Vairetti, M., Ferrigno, A., and Bottiroli, G. (2007) â€Å"Autofluorescence Properties of Rat Liver under Hypermetabolic Conditions.† Photochemical photobiological sciencesà ¢Ã¢â€š ¬Ã‚ ¯: Official journal of the European Photochemistry Association and the European Society for Photobiology 6 (11), 1202–9 Dasari, S. and Bernard Tchounwou, P. (2014) â€Å"Cisplatin in Cancer Therapy: Molecular Mechanisms of Action.† European journal of pharmacology 740, 364–378 Elmore, S. (2007) â€Å"Apoptosis: A Review of Programmed Cell Death.† Toxicologic pathology 35 (4), 495–516 Van Genderen, H.O., Kenis, H., Hofstra, L., Narula, J., and Reutelingsperger, C.P.M. (2008) â€Å"Extracellular Annexin A5: Functions of Phosphatidylserine-Binding and Two-Dimensional Crystallization.† Biochimica et biophysica acta 1783 (6), 953–63 Golstein, P. and Kroemer, G. (2007) â€Å"Cell Death by Necrosis: Towards a Molecular Definition.† Trends in biochemical sciences 32 (1), 37–43 Hatch, E. (2014) â€Å"M19BMS Laboratory Schedule†. Coventry University Kogure, T., Ueno, Y., Iwasaki, T., and Shimosegawa, T. (2004) â€Å"The Efficacy of the Combination Therapy of 5-Fluorouracil, Cisplatin and Leucovorin for Hepatocellular Carcinoma and Its Predictable Factors.† Cancer chemotherapy and pharmacology 53 (4), 296–304 Kroemer, G., Galluzzi, L., Vandenabeele, P., Abrams, J., Alnemri, E.S., Baehrecke, E.H., Blagosklonny, M. V, El-Deiry, W.S., Golstein, P., Green, D.R., Hengartner, M., Knight, R.A., Kumar, S., Lipton, S.A., Malorni, W., Nuà ±ez, G., Peter, M.E., Tschopp, J., Yuan, J., Piacentini, M., Zhivotovsky, B., and Melino, G. (2009) â€Å"Classification of Cell Death: Recommendations of the Nomenclature Committee on Cell Death 2009.† Cell death and differentiation 16 (1), 3–11 Macey, M.G. (2007) Flow Cytometryà ¢Ã¢â€š ¬Ã‚ ¯: Principles and Applications. ed. by Macey, M.G. Humana Press, Inc Maxwell, P. and Hannon-Fletcher, M. (2009) â€Å"Advanced Techniques in Diagnostic Cellular Pathology†. in Advanced Techniques in Diagnostic Cellular Pathology. Hoboken: Wiley, 69–97 Okamura, M., Shimada, J., and Sakagami, H. (2008) â€Å"Comparative Analysis of Cell Death Induction by Cisplatin and 5-FU in Human Oral Squamous and Hepatocellular Carcinoma Cell Lines†. Anticancer research 260, 253–259 Parker, J.B. and Stivers, J.T. (2011) â€Å"Dynamics of Uracil and 5-Fluorouracil in DNA.† Biochemistry 50 (5), 612–7 Rang, H., Dale, M., Ritter, M., Flower, R., and Henderson, G.. (2012) â€Å"Anticancer Drugs†. in Rang and Dale’s Pharmacology. 7th edn. Elesevier, 676–677 Ulukaya, E., Acilan, C., and Yilmaz, Y. (2011) â€Å"Apoptosis: Why and How Does It Occur in Biology?†. Cell biochemistry and function 29 (6), 468–80 Wu, Y., Tibrewal, N., and Birge, R.B. (2006) â€Å"Phosphatidylserine Recognition by Phagocytes: A View to a Kill.† Trends in cell biology 16 (4), 189–97 Appendix Table 1: Table showing mean fluorescence for each sample where 1.00 represents the negative control, 2.00 represents the positive control, 3.00 the untreated sample, 4.00 the sample treated with cisplatin, 5.00 with 5-fluorouracil N Mean Std. Deviation Std. Error 95% Confidence Interval for Mean Minimum Maximum Lower Bound Upper Bound 1.00 3 144.3333 11.93035 6.88799 114.6967 173.9700 136.00 158.00 2.00 3 1866.6667 32.65476 18.85323 1785.5477 1947.7856 1836.00 1901.00 3.00 3 214.3333 10.59874 6.11919 188.0046 240.6621 203.00 224.00 4.00 3 864.0000 20.22375 11.67619 813.7614 914.2386 849.00 887.00 5.00 3 540.6667 51.93586 29.98518 411.6508 669.6825 484.00 586.00 Total 15 726.0000 647.67772 167.22967 367.3280 1084.6720 136.00 1901.00 Table 2 Descriptive statistics where 1.00 is the negative control, 2.00 is the positive control, 3.00 is the untreated sample, 4.00 is the sample treated with cisplatin and 5.00 is the sample treated with 5-fluorouracil. N Mean Std. Deviation Std. Error 95% Confidence Interval for Mean Minimum Maximum Lower Bound Upper Bound 1.00 3 144.3333 11.93035 6.88799 114.6967 173.9700 136.00 158.00 2.00 3 1866.6667 32.65476 18.85323 1785.5477 1947.7856 1836.00 1901.00 3.00 3 214.3333 10.59874 6.11919 188.0046 240.6621 203.00 224.00 4.00 3 864.0000 20.22375 11.67619 813.7614 914.2386 849.00 887.00 5.00 3 540.6667 51.93586 29.98518 411.6508 669.6825 484.00 586.00 Total 15 726.0000 647.67772 167.22967 367.3280 1084.6720 136.00 1901.00 Table 3 ANOVA analysis carried out on the data Sum of Squares df Mean Square F Sig. Between Groups 5863955.333 4 1465988.833 1655.612 .000 Within Groups 8854.667 10 885.467 Total 5872810.000 14 Table 4 Ad-hoc test Tukeys range tests and LSD showing statistical significance between groups. All differences between groups are statistically significant (p Multiple Comparisons Dependent Variable: Fluorescence (I) Treatment (J) Treatment Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval Lower Bound Upper Bound Tukey HSD 1.00 2.00 -1722.33333* 24.29632 .000 -1802.2945 -1642.3722 3.00 -70.00000 24.29632 .094 -149.9612 9.9612 4.00 -719.66667* 24.29632 .000 -799.6278 -639.7055 5.00 -396.33333* 24.29632 .000 -476.2945 -316.3722 2.00 1.00 1722.33333* 24.29632 .000 1642.3722 1802.2945 3.00 1652.33333* 24.29632 .000 1572.3722 1732.2945 4.00 1002.66667* 24.29632 .000 922.7055 1082.6278 5.00 1326.00000* 24.29632 .000 1246.0388 1405.9612 3.00 1.00 70.00000 24.29632 .094 -9.9612 149.9612 2.00 -1652.33333* 24.29632 .000 -1732.2945 -1572.3722 4.00 -649.66667* 24.29632 .000 -729.6278 -569.7055 5.00 -326.33333* 24.29632 .000 -406.2945 -246.3722 4.00 1.00 719.66667* 24.29632 .000 639.7055 799.6278 2.00 -1002.66667* 24.29632 .000 -1082.6278 -922.7055 3.00 649.66667* 24.29632 .000 569.7055 729.6278 5.00 323.33333* 24.29632 .000 243.3722 403.2945 5.00 1.00 396.33333* 24.29632 .000 316.3722 476.2945 2.00 -1326.00000* 24.29632 .000 -1405.9612 -1246.0388 3.00 326.33333* 24.29632 .000 246.3722 406.2945 4.00 -323.33333* 24.29632 .000 -403.2945 -243.3722 LSD 1.00 2.00 -1722.33333* 24.29632 .000 -1776.4689 -1668.1978 3.00 -70.00000* 24.29632 .016 -124.1356 -15.8644 4.00 -719.66667* 24.29632 .000 -773.8022 -665.5311 5.00 -396.33333* 24.29632 .000 -450.4689 -342.1978 2.00 1.00 1722.33333* 24.29632 .000 1668.1978 1776.4689 3.00 1652.33333* 24.29632 .000 1598.1978 1706.4689 4.00 1002.66667* 24.29632 .000 948.5311 1056.8022 5.00 1326.00000* 24.29632 .000 1271.8644 1380.1356 3.00 1.00 70.00000* 24.29632 .016 15.8644 124.1356 2.00 -1652.33333* 24.29632 .000 -1706.4689 -1598.1978 4.00 -649.66667* 24.29632 .000 -703.8022 -595.5311 5.00 -326.33333* 24.29632 .000 -380.4689 -272.1978 4.00 1.00 719.66667* 24.29632 .000 665.5311 773.8022 2.00 -1002.66667* 24.29632 .000 -1056.8022 -948.5311 3.00 649.66667* 24.29632 .000 595.5311 703.8022 5.00 323.33333* 24.29632 .000 269.1978 377.4689 5.00 1.00 396.33333* 24.29632 .000 342.1978 450.4689 2.00 -1326.00000* 24.29632 .000 -1380.1356 -1271.8644 3.00 326.33333* 24.29632 .000 272.1978 380.4689 4.00 -323.33333* 24.29632 .000 -377.4689 -269.1978 *. The mean difference is significant at the 0.05 level.

Learning About Gender Similarities Essay -- Social Issues, Womens Rig

This course, in general, constructs views on psychological issues such as sexuality, the relation between motherhood, ethnicity, race, class, women in the workplace and the roles of women. This course embarks on a variety of theoretical perspectives and concepts to everyday research practices on women. In addition, it focuses on a thought-provoking exploration concerning current research on women’s lives. This essay will focus, largely on my overall experience and understanding about this course, and how it has transformed my life as a whole. Learning about Gender Similarities Accordingly, this course delivers a handful of analyzes on the subject of women. One of the main issues that astonished me in regards to this course is the similarity and the likeness between women and men. We are more alike than we are different. No doubt, there are a few dissimilarities between men and women; however, there are alarming studies in the field of psychology that reveals evidence to such stereotypes and biases to these remarks (Matlin, 2008, p.37). This course examines the variety of subjects in relation to the similarities of men and women, in particularly, gender-role stereotypes, social constructionism and the exploration of women and men in the early historical times to the present (Matlin, 2008, p.41). Before enrolling into this course, my expectations were that men and women are completely different, primarily because culture entitles gender to specific groups in accordance to social constructionist perspective. Being the single girl as well as the youngest in m y family, my expectations to reaching goals were always limited taking into account the social identities that tie into intersectionality. With that said, this course has ... ...tering into this course for the first time, my analysis on females and males was that, we are completely different according to biological, cultural and social views. At this moment, my views on females and males are that we are completely similar. The psychological perspectives on social and personality characteristics, women and work, cognitive abilities and attitudes indeed presents compelling explanation to the comparison of females and males. Moreover, learning about the psychology of women opens my mind to questions and answers further down the road. Taking this course has broadened my understanding in relation to the identity and the role I commit as a woman. In the same way, psychology of women has helped in revolutionizing and transforming women’s lives by means of giving us a sense of empowerment and inspiration to fulfill our needs and aspirations.

Management Essay -- essays research papers

  Ã‚  Ã‚  Ã‚  Ã‚  The Hayakawa family had a son named Mark, who was diagnosed with Down’s syndrome. The family wanted to keep the child but were discouraged and advised not to by doctors. They felt differently about the situation. They felt their child lived on love, which was something they knew they could provide a great deal of. The family was not confident in the experts’ views because they seemed to have a lack of knowledge on the subject and it seemed as though they cared less then they knew about the illness.   Ã‚  Ã‚  Ã‚  Ã‚  The family decided to keep mark at home with them, not institutionalized. He had the freedom to grow and the personal care that is necessary to develop in a healthy situation. The Hayakawas felt that keeping Mark home was a good idea. He not only brightened their lives but their children’s lives as well. Mark could find joy in simple things, which can often reflect onto others. Mark was capable of accepting things as they were more so than most people of â€Å"normal† intelligence. The entire family benefited from the experience of living with Mark by learning to take situations in stride. The family learned patience and tolerance through helping Mark get through each day. Their readiness to deal with Mark carried over into a general method of dealing with people. Mark made them feel special.   Ã‚  Ã‚  Ã‚  Ã‚  The family benefited from not following the directions given to them by the experts. They felt as thou... Management Essay -- essays research papers   Ã‚  Ã‚  Ã‚  Ã‚  The Hayakawa family had a son named Mark, who was diagnosed with Down’s syndrome. The family wanted to keep the child but were discouraged and advised not to by doctors. They felt differently about the situation. They felt their child lived on love, which was something they knew they could provide a great deal of. The family was not confident in the experts’ views because they seemed to have a lack of knowledge on the subject and it seemed as though they cared less then they knew about the illness.   Ã‚  Ã‚  Ã‚  Ã‚  The family decided to keep mark at home with them, not institutionalized. He had the freedom to grow and the personal care that is necessary to develop in a healthy situation. The Hayakawas felt that keeping Mark home was a good idea. He not only brightened their lives but their children’s lives as well. Mark could find joy in simple things, which can often reflect onto others. Mark was capable of accepting things as they were more so than most people of â€Å"normal† intelligence. The entire family benefited from the experience of living with Mark by learning to take situations in stride. The family learned patience and tolerance through helping Mark get through each day. Their readiness to deal with Mark carried over into a general method of dealing with people. Mark made them feel special.   Ã‚  Ã‚  Ã‚  Ã‚  The family benefited from not following the directions given to them by the experts. They felt as thou...

Effects of Migration Into Cities

The effects of moving into a new city or town Today everybody is going in the glamorous world of cities where one can progress and make the best use of his or her life. Parents†¦.. Listen to their talks with their children. They say that my son or daughter will settle or go to abroad one day. And with this they start imagining and forcing their child to get highest degree and run away from where they are. Each parent today has this mentality that there is heaven like something in abroad and much else. There are many such examples to discuss. But for now one is enough I think.In terms of living a life in a new city there are many possibilities of advantages as well as disadvantages as can be seen in education, employment and settlement point of view. These three things can be much better improved in a city where there is much scope to put a step forward towards his or her aim. In a city each one who is settled there has only one intention to earn a gallon of paper notes and coins and live a luxurious and easy way of life. This luxurious life is expected by all people. But there is something called feeling of attachment.There is an attachment towards his or her native place. One cannot forget his or her childhood days or their neighbours and the love between them. Emotions are not allowed in a city or the town. Today since all the places are becoming industrialized there is no space for emotions but only profit. And because of this all the people are running after them. There is a great effect on especially the country. Since all the citizens are settled in abroad and the income they earn is of no use to the country, the country’s economic status lowers as compared to other countries.The newer generation is no longer staying in their native country specially India. India is full of corruption, they say. But it is not so. If the abroad based thinkers try, the situation can be changed. The major disadvantage of moving to a new city is the loss of attach ment to the native place and their neighbours. From birth we are attached to our villages or town, its nature, its culture and its people. But as we move to a new area, far from ours, we cannot accept its culture and people very easily. We cannot adjust there due to our unforgettable habits with our ulture. As we move to a new city, we have to accept the people, the culture, and their behaviors and accordingly we have to adjust. But it is not so easy. Since the people are keenly interested in earning money and luxurious life they are ready to do anything for this. According to me, I can’t accept the concept of moving to a new place permanently. Because it affects our native place or country. Earlier during the British rule all the intelligent brains of India were sent out to England or the other countries out of India.So that the foreign countries would progress better in terms of technology. But with this, India had to suffer in its progression. Today after British rule is n o longer still their mentality always exists in each Indian that to go to abroad but not to stay here in India. The aim of writing this article is to aware the people of not to go to abroad but it is better and much better to stay in your own country and sacrifice your talents to your people and their future. – Prachiti Ganpule SYBA, St. Xavier’s college, Mapusa Goa