Cell Culture and Proliferations | Experiment

Cell Culture and Proliferations | Experiment To study the cell proliferation of Mouse Y1 adrenocotical cells by using MTT (3-(4, 5-diphenyl tetrazolium bromide) and crystal violet staining methods. INTRODUCTION In general, Cell proliferation means multiplication/Reproduction of cells for increasing cell population in a very short span of time. The assay of cell proliferation is to measure the number of cells which are present in the dividing culture medium. Cell proliferation is controlled by using growth factor (Fetal calf serum), Which normally bind to the surface receptors present on the cell membrane generally regulate the cell signaling molecules, which pass the message to nucleus by help of receptor generally where the transcription factor gets bind to the DNA, makes turn off turn on the protein synthesis mechanism, responsible for cell division. Cell proliferation method is very important for studying various biological factors like bioassay, carcinogenic analysis and other toxicological tests. Generally two metods are used for studying cell proliferation i.e. crystal violet staining method and MTT (3-(4, 5-diphenyl tetrazolium bromide) method, here these methods are used to study ce ll growth in mouse Y1 adrenocotical cells. In crystal violet staining method, the DNA of cells is going to stained by crystal violet which produces a colour intensity that is proportional to the cultured cells(including newly proliferated cells). In this method, the principle involved to calculate the cell proliferation is based on the absorbance taken up by the viable cells in culture at different concentrations after the cells are stained with crystal violet. In MTT (3-(4, 5-diphenyl tetrazolium bromide) method, the assay depends on the amount of MTT taken up by the cells, tetrazolium salt is water soluble which produces yellow colour. The tetrazolium MTT is metabolically reduced by active cells, in presence of dehydrogenase enzymes, producing NADH and NADPH which are reducing equivalents. This results in the formation of purple formazan intracellularly, which is measured by spectrophotometer. MATERIALS METHODS Cell culture: In DMEM (Dulbeccos modification of eagles medium),mouse Y1 adrenocotical cells which were grown on monolayer was removed by using mixture of trypsin and EDTA (0.05% and 0.02% ).The cells are incubated by 5minutes by adding Trypsin/EDTA(7ml).After incubation the flask was removed and tapped gently to separate undetached cells. The contents of the cell are transferred to a universal container for centrifugation at 1000rpm/ 5mins.Supernatant was discarded and medium is added for resuspending the cell pellet. The cell number was estimated by using Haemocytometer counter for 15ml the cell suspension dilution was prepared containing density 1.25X105 cells/ml of suspension. In 96 well plate, 60 wells were filled with 100Â µl cell suspension, in this the remaining outer wells are filled with Phosphate Buffer Solution (PBS) of 200Â µl.Allowed the plate overnight in a gas incubator to settle down the cells. Those cells were treated with Fetal Calf Serum (FCS) of different concentrations vary ing from 0% to20% in universal tubes.12 wells of plate were filled with 200Â µl of different concentrations. These plates were incubated for a period of 24hours. Cells were washed with Phosphate Buffered Solution (PBS) for three times using multichannel micropipette; later media containing various serum concentrations were added. These two 96 well plates were used for crystal violet staining and MTT (3-(4, 5-diphenyl tetrazolium bromide) assay after 72 hours of incubation. Crystal Violet Staining Method: The cells were removed from central 60 wells of 96 well plates and filled with 200Â µl of methanol in a fume cupboard. After 15minutes methanol was removed from the plate and was left to dry in the fume cupboard. Once the plates were dried the cells were stained with 200Â µl of crystal violet. Once the staining is complete after 20minutes the plates were washed with Distilled water for atleast three (3) times and then solubilized the cell layer by using 50Â µl of 10% glacial acetic acid. The plates were then kept for incubation in a gas incubator for thirty minutes after incubation Absorbance of wells was measured at 540nm. MTT STAINING METHOD: The cells present in the central 60 wells of 96 well plate were treated with 20Â µl of MTT (5mg/ml solution in PBS) and was left for 4hours in gas incubator at 370c.After incubation by using multichannel pipette medium was removed and 100Â µl of acid-isopropanol was added in order to dissolve the blue formazan crystals from the cell layer and then it was incubated for 30minutes at room temperature after solubilizing formazan crystals Absorbance was measured at 570nm using plate reader. Calculation: Total No. of cells in 5 square = 21 Average cells present in one square = 4.2 Calculation of cell number: The volume of each square is= 4 X 10-3 The total cell number for 5 square gives the cell = 0.02Â µl No. of cells in 1 ml = 105X104 Number of cells required = 395X104 Volume of suspension required = concentration required/ concentration got = 395X104/105X104 = 3.7619 ml cell suspension to be taken Medium to be taken =30,000Â µl-3.7619Â µl = 29996.23Â µl medium to be taken. DISCUSSION: Here the increase in the absorbance with corresponding to the fetal serum concentrations shows the sign for the cell growth. Ammonium cations bind to negatively charged DNA which in turn gave blue color to the mixture. By using the color intensity, viable cells were estimated by means of haemocytometer. No experiment will produce 100% results. So here also errors occurred due to practical errors. Occurrence of errors might be due to: Washout condition of stained culture cells Improper solubilisation of 10% glacial acetic acid. In MTT method the degradation of MTT gives color to the mixture. This degradation was due to the dehaydrogenases of viable cells. The color intensity is directly proportional to the cell growth. Here also the errors might be occurred due to improper solubilisation of formazan crystals (Butler. 1996), (Javoise. 1998). DIFFERENTIATION OF K562 CELLS TO PLATELETS IN PRESENCE OF PMA: Differentiation of K562 cells to megakaryoctes/platelets Phorbol Myristate Acetate treated and untreated cells were spun down in a bench centrifuge and after resuspended in 1ml PBS (having 1 % Bovine serum albumin). Then by using haemocytometer the cell number was calculated, after diluting the suspension. cytospin was added to 1 ml cell volume which was adjusted to density of 106 cells per ml. In assembled cytospin 200 Â µl at 1000 rpm/3min. After fixing the slide in acetone /methanol(50:50), slide was washed with 0.15 M tris buffered saline .In humid temperature Human cd61 cells were incubated for 2 hrs using TBS slide was washed.with rabbit anti- mouse Ig-G anti body cells incubated for for 30 mins at room temperature and washed with TBS, after washing, cells were incubated with Alkaline phosphate anti-alkaline phosphate complex, this was repeated with Ram and APAP for amplification. They were washed under running tap water after staining with red TR substrate and counter was stained with haemotoxylin. Finally the slide was viewed und er microscope after washing with TBS.PMA is a diester of phorbol and a tumor promoting agent (proc.Natl.Acad.sci.USA Vol.82, pp, 3859-3862, june 1985Medical sciences). PMA initiates the signal transduction by protein kinase C (PKC) enzyme which allows promoting the differentiation of K562 cells.By using CD 61 marker the K562 cells were treated .These CD61gets attached to cell network to work as primary antibodies. In addition with cells performs a seconndary antibobodies whenever exposed to Ram along with APAP and forms pink color by attaching to FC region of anti-human CD 61 antibodies. This phenomenon gives the cells under going differentiation when incubated with PMA (MSc Pharmacology Biotechnology, cell biology laboratory manual/ January 2010) RESULT: The slide treated with Phorbol Myristate acetate (PMA) is in pink colour, whereas the slide which is untreated with Phorbol Myristate acetate (PMA) is in blue colour after staining. The cells when treated with PMA differentiate into Platelets/Megakaryocytes. In PMA the diseter bond promotes the tumor, which in turn activates the signal transduction of protein kinase C enzyme(PKC) inn K562 cells causes the CD61 expression. The RAM IgG gets attached to the CD61 antibodies, these K562 cells when incubated with APAAP form a complex. Later fast red dye was added to the mixture which gets attached to the APAAP, the cells turn pink by taking the stain. The cells containing PMA expressed the CD61. It generates the (signal transduction protein kinase C) PKC enzyme and on of K562 cells causes the expression of CD61. The rabbit antimouse IgG antibodies attach to the antibodies of CD61 when incubated in the presence of APAAP (alkaline phosphatase anti-alkaline phosphatise) on of K562 cells complex. Then we add the fast red dye to the mixture which was attached to APAAP (alkaline phosphatase anti-alkaline phosphatise) and stains the cells pink. CD61 was expressed by only those cells which had PMA (Shelly, 2000)

Drama Coursework Essay

The main reason I chose this extract was that there were two female characters in the extract and two female actors in the group. I liked the way the relationship between the two characters developed during the extract. At the beginning of the play, there is a professional, quite friendly relationship but by the end, both characters hate each other. I also liked the way my character, Mrs Lyons, descends into madness. When I first started to rehearse this play, I found that it was harder than I expected. This is the first serious play I have been involved in, so I found it hard to say the lines convincingly and naturally. I suggested that I should talk with a more educated voice and that my partner, Becky, should talk with a more â€Å"common† voice to emphasise the social difference between the two characters. I also suggested some ideas for what we should wear. In my coursework I wanted to present a convincing portrayal of a wealthy, educated woman who, faced with a extremely distressing, seemingly insurmountable personal problem, sees what looks like a perfect solution, but which ends up driving her to madness. I wanted to initially gain the audience’s sympathy for her predicament by showing how much she wanted children and to show her as a reasonable person. I considered the second scene the most important as this was when my character discovered that her employee was expecting twins and couldn’t afford to keep them both; and when the plan for Mrs Lyons to pretend to be pregnant and to keep one of the babies was hatched. In this scene, she promises Mrs Johnstone that the baby will be better off with her, and that Mrs Johnstone will be able to see him every day as she comes to work. However, in the next scene, she breaks that promise by sacking her. I wanted to show that the sacking was motivated by Mrs Johnstone’s paranoia. In the final scene I wanted to demonstrate that my character’s mental health had deteriorated. I tried to portray that she was wealthy and educated was by talking in an upper-class, educated accent, and by dressing in a smart suit. I could have improved my performance by making my accent more pronounced, doing my hair in a more sophisticated way, and wearing some tasteful make-up. In the first scene I tried to convey her longing for a child by delivering the words as if I was completely wrapped up in my problem and as if I was talking to myself rather than anyone in particular. To demonstrate this, when I said the lines about only buying such a big house in the hope of having children, I looked down at the table rather than looking at my partner because I wanted to make it seem as if Mrs Lyons felt vulnerable because she was being so open. I concentrated on what I was doing at the time, which was getting something out of my bag, to try and convey that my character was fighting back tears, and didn’t want to look at Mrs Johnstone in case she showed her any sympathy or pity, which might have made her cry. I think I could have emphasised my character’s reaction when she found out that Mrs Johnstone was pregnant in a similar way to Kara when she choked back her tea. In the second scene I wanted to show the first signs of my character’s madness, when, on learning that Mrs Johnstone is expecting twins, she comes up with the extraordinary idea that she should fake her pregnancy and take one of the babies. I tried to express how she got more excited by speaking more quickly and by the tone of my voice. I also moved around a lot and started to talk more to myself than to my partner as my character got more carried away with her plan. In the third scene, where Mrs Lyons sacks Mrs Johnstone, I started off talking in a very authoritative tone and avoided eye contact with Becky because the supply teacher explained that when you have a problem with someone, you don’t look at them. As the conversation develops, and Mrs Johnstone threatens to take the baby away or tell the police, I wanted to show that Mrs Lyons was manipulating Mrs Johnstone by playing on her superstition and lying to her that she’ll be locked up if she tells anyone what happens. I showed this by getting close up to Becky and talking quite slowly and with a deep voice, in a threatening way. Because I am quite a bit taller than Becky, I was also able to look down on her, which reflected the difference in class between the two characters. I think it was a mistake to avoid mannerisms as compared to some other groups, whose little individual touches made their plays stand out, our performance was quite boring.

Workplace, Childcare And Early Childhood Education Essay

Question 2 Setting: Workplace, childcare and early childhood education Some mothers may return to work early after giving birth for many reasons such as financial strains (Abdulwadud Snow, 2012). Workplaces need to support women who return to work after breastfeeding to ensure they are able to maintain breastfeeding for the recommended duration (Abdulwadud Snow, 2012). A study by Galtry found that countries that have established initiatives for paid maternity/parental leave, have longer maternity/parental leave entitlements, have introduced breastfeeding breaks in all workplaces, and have developed policies that require employers to support breastfeeding, have higher exclusive breastfeeding rates than other countries (Galtry, 2003). 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How Social Media is Effecting Social and Communication Skills Among Adolescents - Free Essay Example

Sample details Pages: 2 Words: 595 Downloads: 7 Date added: 2019/04/10 Category Society Essay Level High school Tags: Social Media Essay Did you like this example? Social media has taken over everything especially on real life communication. To begin with, the way people communicate on social is not the same way as before. Internet based life and online correspondence is accepted to effectually affect social abilities and correspondence among youths. Don’t waste time! Our writers will create an original "How Social Media is Effecting Social and Communication Skills Among Adolescents" essay for you Create order In the no so distant past online networking did not exist and social correspondence and cooperation were the main method for imparting. Web-based social networking likewise makes a picture for individuals based off their profiles, posts and pictures. Be that as it may, today individuals can convey and banter through web based life from anyplace on the planet. This is accepted to frustrate societys social aptitudes and correspondence, and even reason emotional wellness issue. A standout among the most detectable impacts of internet based life on society is social correspondence. Social aptitudes and communication are credits individuals need to prevail throughout everyday life. It might be difficult to see, however kids manufacture these aptitudes as they grow up communicating with other kids in up close and personal situations. Online life is extremely restricting associations amongst kids, and above all, amid a pivotal time in their lives. Whenever kids and teenagers are youthful is the time when social aptitudes are being constructed. Albeit internet based life can adversely affect immatures social abilities, it might really be emphatically affecting connections among peers. High school years are regularly intense for some. It is a period of stress, learning, and perplexity. Solid connections among companions are the way most youths adapt to pressure. The utilization of online networking and being able to stay associated with companions may emphatically affect connections among peers. On the off chance that internet based life is utilized effectively as for this situation of saving connections, youths social aptitudes ought not to decrease to such an extent. Having solid associations with companions may simply change issues with social abilities among youths. A negative segment that accompanies online networking is digital tormenting, and it can be a noteworthy reason for the abatement in social abilities among young people. Internet based life considers young people to get in touch with each other at any snapshot of the day. At the point when an understudy who is tormented throughout the day at school, they are soothed to return home to get away from the antagonism and pitilessness. Nonetheless, web based life has made it about difficult to get away from the cruel words and mean remarks of others. Always being tormented will effectually affect a juveniles confidence and certainty. It will make youngsters apprehensive to interface in school, get associated with games, and general abatement their social inclusion. Social and relational abilities among teenagers are critical aptitudes that are encountering both negative and constructive outcomes from web based life. There are conspicuous parts of online networking that are hurting social aptitudes, for example, spending various hours on Instagram, and after that there are unpretentious angles, for example, the like component. There is nothing unexpected that web based life is digging in for the long haul with its accessibility and value in the public arena today. Clearly web based life is a noteworthy segment of ad for organizations and correspondence for individuals around the globe, and this is totally adequate. Be that as it may, the line must be drawn when internet based life is seen devastating youthful social aptitudes, correspondence, and causing hostile to social conduct. In the event that web based life is utilized effectively, for example, keeping up connections among peers, social abilities and correspondence for young people ought t o stay unblemished.