Culture Of Hepg2 Cells Biology Essay

Culture Of Hepg2 Cells Biology Essay Hep G2 cell line was purchased from American Type Culture Collection (ACTT) (VA, USA). Dulbeccos Modified Eagle Medium (DMEM), 0.5% Trypsin-EDTA 10x, and Penicillin-Streptomycin (PS) were obtained from Invitrogen Corporation (NY, USA). Fetal Bovine Serum (FBS) was gotten from Welgene Inc. (Daegu, South Korea). Fatty acids (Palmitic, Oleic and Dedocanoic acid), Dimethyl sulfoxide (DMSO) and Tween 20 came from Sigma (MO, USA). Bovine serum albumin (BSA) was from Santa Cruz Biotechnology (CA, USA). MTT assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) was purchased from Molecular Probes (Oregon, USA). LDH assay (Lactate dehydrogenase assay) was from ROCHE (Mannhein, Germany). [emailprotected]/503 and Carboxyl-H2DCFDA were purchased from Invitrogen Corporation (Oregon, USA). Nile red was from Fluka (MO, USA). Triglyceride Quantification Kit and ATP Colorimetric/Flourometric Assay Kit were purchased from BioVion Inc. (CA, USA). Annexin V Floustaining kit was from Roche (IN, USA). Phosphate buffered saline was made up of chemicals at pH 7.4, including 11.7g NaCl, 5.5g Na2HPO4-7H2O, and 1.35g NaH2PO4. All other chemicals met in standard grade of analysis. Culture of HepG2 cells HepG2 cells were cultured in Dulbeccos modified Eagles medium, containing 10% (v/v) fetal bovine serum and 1% (v/v) Penicillin-Streptomycin under 5 % CO2, 95 % humidity at 37 °C. The cells were subcultured by using 0.5% Trypsin-EDTA 1x (Invitrogen Corporation, NY, USA) for detachment and seeded at proper cell number in all experiments. Fatty acid treatment When 80 % confluency of HepG2 was reached, it was treated with various concentrations of the fatty acids (0 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.5 mM, 0.7 mM and 1.0 mM) for 24 h. The stock solution of fatty acids was prepared at 100 mM by dissolving in DMSO and stored at -200C. The stocks were diluted in DMEM media containing a constant ratio of fatty acid bound bovine serum albumin at 2 to 1 to obtain working solution in all experiments. Cytotoxicity assay Cytotoxicity was based on the measurement of cytoplasmic enzyme activity by using cytotoxicity detection kit (ROCHE, Mannhein, Germany). The cytoplasmic enzyme was released from damaged cells that its enzyme activity expresses to the proportion of toxiced-cell. Lactate dehydrogenase (LDH) presents in all cells which is a stable cytoplasmic enzyme. When the membrane integrity of the cells is damaged, it is quickly released into the media. In this assay, NAD+ is reduced to NADH/H+ during conversion of lactate to pyruvate by the LDH-catalyzed. After that, H/H+ from NADH/H+ was transferred by the catalyst (diaphorase) to the tetrazolium salt (yellow) which was reduced to formazan (red). To conduct the assay, the culture supernatant is collected cell-free after desire exposure time (24 h). The reaction mixture from the kit was then applied in the samples. The absorption of the formazan dye formed was measured at 490 nm on an ELISA reader (VERSARMAX, Molecular Divices., CA, USA). Cell viability Cell viability was measured based on measurement absorption of a water-insoluble purple formazan which was reduced from a yellow water-soluble tetrazolium salt in live cells. Briefly, the cells were treated with MTT (5 mg/ml) in DMEM at 37 0C for 1.5 h. Then, the media were removed, and DMSO was added to dissolve the furmazan crystals. After gently pipetting, the absorbency was measured at 570 nm using an ELISA reader (VERSARMAX, Molecular Divices., CA, USA). The estimation of cell viability was calculated by comparing between the spectra value of treated and untreated cells. Quantification of triglyceride Triglyceride content (TG) was determined according to an enzymic method (BioVion Inc, CA, USA). In this method, glycerol is a product by TG-catalyzed which reacts with the probe to generate coloration measured on spectrophotometry at 570 nm. In briefly, the cells were washed twice times with cold PBS, then homogenized in 5% Triton-X100 solution. After slowly heating at 80-100 °C for 5 min, the samples were centrifuged at 12000 rpm for 5 min. The supernatant collected from removing insoluble materials was added 2 ÃŽ ¼l of lipase, mixed well and incubated for 20 min at room temperature. Finally, 50 ÃŽ ¼l of the reaction mix was putted in each sample for 45 min of incubation, protected from light. The value of triglyceride content was quantified based on triglyceride standard curve that was constructed with different concentrations of TG (0, 20, 40, 60, 80, and 100 nmol/ml). Measurement of reactive oxygen species (ROS) generation The measurement of ROS production within cells was carried out by using 2†²,7†²-Dichlorohydrofluorescein diacetate (Carboxyl-H2DCFDA; Invitrogen Corporation, Oregon, USA) which is combined into fluorescent products in the presence of H2O2 and other ROS molecules and esterases (Zhenyuan Song et al, 2007). After the cells were overloaded with 1.0 mM fatty acids, 10 mM final concentration of Carboxyl-H2DCFDA was added in the media without FBS at 370C in darkness for 30 min. Then, the cells were washed twice times with warmed PBS and lysed in 200 Ã‚ ­l RIPA buffer (PIERCE, IL, USA). The lysed-cells were centrifuged at 12000 rpm for 5 min. The supernatants were conveyed to a 96-well back plate which were excited at 485 nm and emitted at 530 nm for the Carboxyl-H2DCFDA fluorescence on Fluorometer (VICTOR2, Perkin Elmer., MA, USA). Trilyceride staining on Confocal Bodipy @493/503 (Invitrogen, Oregon, USA) was used to capture TG fluorescence on Confocal microscopy. In this experiment, the cells were prepared as above. Before the dyes treatment, the cells were washed with PBS twice times. Bodipy @493/503 was then added at 1.0  Ã‚ ­M, and 15 min of incubation at 370C after the cells were rinsed with PBS again. Zeiss LSM Image Brown software (LSM 510 meta, Carl Zeiss., Jena, Germany) was handled to take TG image at excitation of 488 and emission of BP 505-530 nm. ROS staining on Confocal ROS generation in HepG2 was stained by using Carboxyl-H2DCFDA. In this experiment, the cells were prepared as above. Before the dyes treatment, the cells were washed with warmed PBS twice times. The carboxyl-H2DCFDA was applied at 10 mM final concentration in Serum free media (DMEM without FBS), and incubated for 30 min at 370C, protected from the light. After that, the cells were rinsed with warmed PBS again. Zeiss LSM Image Brown software (LSM 510 meta, Carl Zeiss., Jena, Germany) was handled to take ROS image at excitation of 488 nm and emission of LP 530 nm. Detection of cell death and trilyceride accumulation by Confocal HepG2 seeded in the 24-well plate and treated with final concentration of fatty acids to 1.0 mM for 24 h. After the incubation time, the cells were washed twice times with PBS. Then, Bodipy @493/503 (Invitrogen, Oregon, USA) was dissolved in PBS at 5  Ã‚ ­g/ml which was added into each well. This process was kept in darkness for 15 min at 370C. After that, the Bodipy solution was removed and the cells were then washed by Binding buffer from Annexin V Floustaining kit (Roche, IN, USA). Finally, the cells were incubated in 100  Ã‚ ­l/ml of Propidium iodide (PI) for 10 min in darkness. Exposition of TG accumulation and apoptosis was observed at excitation of 488 and 543 nm, and emission of BP 505-530 and LP 650 nm on Carl Zeiss Confocal Microscopy (LSM 510 meta, Carl Zeiss., Jena, Germany), respectively. Data analysis All results were expressed as mean of repeated three or four values  ± SEM. The difference between groups was identified by using t.test. p < 0.05 was considered statistical significant.

Patient Interview Essay

There are many components to consider a patient interview to be effective. During the workshop week in Toronto, I have learned those basic yet very essential components through the enactment presented. Firstly, it is really important to establish a good rapport when dealing with patients. A good rapport can create a relationship that is built on trust and commitment. Through this, patient can share private medical information without hesitations. An example of this was when the pharmacist greeted the patient and asked how can she be of help. She also showed empathy when she found out that the patient was in pain and told the patient she’d prepare the prescription right away. Listening is also an essential component. As a health care provider, listening gives the opportunity to know their needs and concerns. Acknowledging what the patient is really saying, maintaining eye contact, and recognizing and using body language are some of the things that I need to consider. If these are all effectively met, I think that this would help patients in becoming more involved with their medications/treatments, thus producing a positive patient outcomes. The probing or the way I ask question to patients also plays a vital role. It is important that I ask questions in a sincere way to obtain needed information or to just simply clarify something. Asking open-ended questions will help elicit relevant answers from the patient and not just â€Å"yes/no† answers. An example would be when she asked for allergies and asked for the specific kind of reaction that she had. Lastly, feedback is a must. Before ending the interview, asking for a feedback will allow me to check if the patient really understands what was taught. . An example of this was when she asked the patient how she would be taking the medication. This will help to reinforce adherence and make the patient to be involved in the treatment, reducing or eliminating chances of non-compliance. Patient interview is very important for a positive treatment outcome. It is not a simple process but I do hope that as I go along, I would be able to utilize all these components and achieve an effective patient interview.

Love and Relationships Updates

Communication is the key to maintaining a healthy relationship. Taking serves to nurture your relationship and prevents problems or issues from festering. † Working things out is part of every relationship and everyone will do it differently. Many would have never been that serious when it comes to knowing more about how relationships work and how will it ever go well. Here’s how to deal with conflicts and how to manage your relationships. Learn to manage your time. If you really love the person you are in a relationship with, you should never forget that time is very much important when it comes to relationships. Remember that some people feel important when they are given time. To spend time on something thoroughly and effectively, takes time. And while this sounds redundant, it is a fundamental truth: to take time, takes time. Therefore, knowing how to use time effectively is essential. Use healthy communication to resolve conflicts. Try to see things from each other’s point of view. Negotiate in times of disagreements; understand that you cannot win at all times. Listen without judging. Stick to issues and do not attack the person, his beliefs or even his culture. Accept each other’s uniqueness. Realize that your differences enrich your relationship. Don’t sweat the small stuffs out. â€Å"Accept my supposed quirkiness as a woman and I’ll be accepting yours as a man. † as others would usually put in. From the start, build a foundation based on respect and apprec- iation of each other’s characteristics. Explore each other’s differences and interests and build them to make your relationship as a couple stronger. Don’t drag about the past. You see, PAST is PAST. You should never go back to it anymore because it is done. All you have to do is just to learn from it and prevent your mistakes and continue doing what is simply right. Take time to reflect on your own history as a third party looking in without judgment: simply observe. Understand that you are not your past. Understand that the situations and patterns and people in your life created your experiences, they didn’t create you. Knowing and understanding your past and some of your patterns will help you to recognize why you hold on and repeat self-destructive behaviors. Understanding creates awareness; awareness helps you break the cycle. Build your trust. Trust is the treasure of our daily lives. However, we do not understand its value. It is generally seen that trust in our daily lives is disappearing fast. Why have we become so suspicious that we can never enter into meaningful relationships with each other? Why can we not behave as normal human beings? After all when we were born as human beings the first lesson we learnt was that we should trust each other. However, as our lives progressed slowly, trust began to diminish. Our childhood innocence gave way to calculations in which there was no place for trust. Trust in each other gives strength and vitality to our relationships. It gives us inner happiness, which is priceless. It brings joy all around and life appears brighter and brighter . Its fragrance spreads far and wide. When you trust each other you feel self-confident. Trusting each other gives us a sense of deep bonding. It signifies that we are united to fight the battles ahead. It is indeed the communication in which relationships rely into. Without proper understanding and communication, a relationship can either deteriorate or be simply gone in a blink. The reason why most relationships couldn’t gone farther and longer is because they cannot talk about their problems, and egoistic people always are egoistic. Nobody wants to go under the other. Love is never about one but is about two or more hearts bind together as ONE. Love thinks less of oneself and it always gives and protects the other. Whenever we think that we are going wrong in love, we should never forget of the definition or should we say real definition of love in the Bible, it’s I Corinthians 13.

Davis Home Project Privacy Fence - 1444 Words

Davis Home Project: Privacy Fence The Davises recently purchased a new family home in a subdivision. This is a completely new environment for them since they are used to living in the country with room and privacy to spare, and also no neighbors. In order to feel more secluded, they have decided to add a privacy fence around their back yard. The goal of the privacy fence project is to provide the Davis family privacy, but also to clearly define the property line between their yard and the adjoining neighbor’s yard. Mr. Davis is very handy, and generally completes all of the home projects himself with the supervision of the very particular Mrs. Davis. Mr. Davis has also acquired an impressive collection of power and hand tools over the years, so the ones that are needed to complete this project have already been procured. Mr. and Mrs. Davis have created a budget and a timeline for the project, and have agreed to spend no more than $1000. Since Mr. Davis is using some of his vacation days to complete this project, there will be a strict 5-day timeline, starting on Monday, July 6, 2015 and ending on Friday, July 10, 2015. Measure of Project Success and Failure According to Kloppenborg (2015), â€Å"Project success is creating deliverables that include all of the agreed-upon features† (p.11). Other measures of success include the customer’s success, the performing organization’s success, the project team’s success, and if the project is completed on time and on budget.Show MoreRelatedCloud Computing Security67046 Words   |  269 Pagessecurity standards globally, aligning multiple, disparate government policies on cloud security and putting forward standards for ratification by international standards bodies. CSA sees itself as a cloud security standards incubator, so its research projects use rapid development techniques to produce fast results. To this end, the CSA Guidance editorial team is proud to present the third version of its flagship â€Å"Security Guidance for Critical Areas of Focus in Cloud Computing.† This work is a set ofRead MoreEssay on Fall of As clepius95354 Words   |  382 PagesDuncan! a petite girls voice called out. Carmine! The Latino girl jumped up and grabbed Duncan. They locked lips and held it for a minute. She backed her head away and held Duncan by his chin. Hey there, she cooed. Did you finish that project yesterday? Yes, Carmity. I finished it. Did you? Carmine was a beautiful woman who Duncan was dating. She had milky chocolate skin and lush brown hair. Carmine always had a positive attitude and was very intelligent, but she is very arrogantRead MoreHuman Resources Management150900 Words   |  604 PagesFastest Employment Growth, 1996—2006 Numbers in Thousands of Jobs Occupation Database administrators, computer support specialists, and all other computer scientists Computer engineers Systems analysts Personal and home care aides Physical and corrective therapy assistants and aides Home health aides Medical assistants Desktop publishing specialists Physical therapists Occupational therapy assistants and aides Employment 1996 212 216 506 202 84 495 225 30 115 16 2006 461 451 1,025 374 151 873 391 53Read MoreDeveloping Management Skills404131 Words   |  1617 PagesAcquisitions Editor: Kim Norbuta Editorial Project Manager: Claudia Fernandes Director of Marketing: Patrice Lumumba Jones Marketing Manager: Nikki Ayana Jones Senior Marketing Assistant: Ian Gold Senior Managing Editor: Judy Leale Senior Production Project Manager: Kelly Warsak Senior Operations Supervisor: Arnold Vila Operations Specialist: Ilene Kahn Senior Art Director: Janet Slowik Interior Design: Suzanne Duda and Michael Fruhbeis Permissions Project Manager: Shannon Barbe Manager, Cover VisualRead MoreLogical Reasoning189930 Words   |  760 Pagesadvice, etc. Dowden gets the balance and the emphasis right. Norman Swartz, Simon Fraser University v Acknowledgments For the 1993 edition: The following friends and colleagues deserve thanks for their help and encouragement with this project: Clifford Anderson, Hellan Roth Dowden, Louise Dowden, Robert Foreman, Richard Gould, Kenneth King, Marjorie Lee, Elizabeth Perry, Heidi Wackerli, Perry Weddle, Tiffany Whetstone, and the following reviewers: David Adams, California State PolytechnicRead More_x000C_Introduction to Statistics and Data Analysis355457 Words   |  1422 PagesOlsen, Jay Devore Acquisitions Editor: Carolyn Crockett Development Editor: Danielle Derbenti Assistant Editor: Beth Gershman Editorial Assistant: Ashley Summers Technology Project Manager: Colin Blake Marketing Manager: Joe Rogove Marketing Assistant: Jennifer Liang Marketing Communications Manager: Jessica Perry Project Manager, Editorial Production: Jennifer Risden Creative Director: Rob Hugel Art Director: Vernon Boes Print Buyer: Karen Hunt Permissions Editor: Isabel Alves Production Service: