1. Cell Preparation and Treatment<ol><li>In a 24-well plate, put a glass cover at the bottom of the wells. Wash each well with phosphate-buffered saline (PBS).</li><li>Seed Huh-7 cells supporting hepatitis C virus (HCV) subgenomic replicon in each well to a density of 5 &#xD7; 104 cells/well. If there is no treatment to do, the cells can be seeded at a higher density. The culture medium is Dulbecco&#x2019;s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 2mM L-glutamine, 1 mM sodium pyruvate, and 250 mg/ml G-418 (to maintain the replicon).</li><li>Incubate at 37 &#xB0;C and 5% CO&#x2082;. The next day, treat the cells with 3 &#xB5;M of human lactoferrin (hLF) purified from human milk.</li></ol>2. Paraformaldehyde/Sucrose Preparation (12% PFA and 12% sucrose)<ol><li>Put 500 ml of PBS in a beaker and heat it between 20 &#xB0;C and 30 &#xB0;C. Dissolve 60 g of sucrose in the PBS. Dissolve 60 g of paraformaldehyde (PFA) in the PBS/sucrose solution. Slowly add sodium hydroxide 2N (3 to 7 ml) until a clear solution is obtained.</li><li>Adjust pH to 7.4 with hydrochloric acid. Complete volume to 500 ml with PBS. Filter by gravity on a Whatman filter. Before use, dilute the solution with PBS to a final concentration of 4% PFA and 4% sucrose (Storage 4 &#xB0;C for 1 week maximum).</li><li>At the desired time (0 hr, 2 hr, or 24 hr of treatment), wash cells twice (1 min) with PBS and fix with PBS/4% PFA/4% sucrose for 20 min at room temperature. The cells are typically at 30-40% confluence, and 1.5 &#xD7; 105 cells are used.</li></ol>

<table class="ck-table-resized"><tbody><tr><td>Dulbecco&#x2019;s Modified Eagle Medium (DMEM)</td><td>Wisent</td><td>319-005-CL</td><td>&#xA0;</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>BioShop</td><td>PAR070.1</td><td>Flammable solid, skin irritant, lungs and eyes</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Wisent</td><td>311-425-CL</td><td>Without calcium &amp; magnesium ions</td></tr><tr><td>Human lactoferrin (hLF)</td><td>Sigma</td><td>L0520</td><td>&#xA0;</td></tr></tbody></table>

preparing cells to analyze intracellular uptake and activity of an antiviral agent

Preparing Cells to Analyze Intracellular Uptake and Activity of an Antiviral Agent

Source:<a href="https://app.jove.com/v/53053/immunofluorescence-to-monitor-the-cellular-uptake-of-human-lactoferrin-and-its-associated-antiviral-activity-against-the-hepatitis-c-virus"> Allaire, A., et al. Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus. J. Vis. Exp. (2015).</a>The video demonstrates the procedure for preparing cells to assess the intracellular uptake and activity of an antiviral agent against the hepatitis C virus.

https://cloudfront.jove.com/files/ftp_upload/24553/24553_article_thumb_24553.jpg

Begin with human hepatoma cells cultured on coverslips in a multi-well plate.These cells contain the hepatitis C virus genome in the cytoplasm, which produces viral proteins essential for viral genome replication.In the control well, maintain the cells in culture medium alone, while in the test well, add lactoferrin, an antiviral agent.Incubate the plate to allow lactoferrin to enter the cells through endocytosis and accumulate in the cytoplasm.Inside the cytoplasm, lactoferrin disrupts the viral proteins, halting further viral genome replication.In the control well, the viral genome replicates into negative-sense RNA intermediates, which are then used to produce positive-sense RNA copies that serve as templates for viral protein synthesis.After treatment, wash the cells with a buffer to remove unbound lactoferrin.Add a fixative solution with sucrose. Incubate to fix and stabilize the cells.The cells are now ready for the analysis of intracellular uptake and antiviral activity of the treatment agent.To begin, place a round cover glass in the bottom of each well of a 24-well plate and use PBS to wash the wells. Seed HUH seven cells carrying the HCV Replicon at a density of five times 10 of the fourth cells per well in DMEM supplemented with 10% FBS, two millimolar L-glutamine, one millimolar sodium pyruvate, and 250 milligrams per milliliter G-418. Incubate the cells at 37 degrees Celsius and 5% carbon dioxide overnight.The following day, treat the cells with three micromolar human lactoferrin or HLF purified from human milk and incubate for 0, 2, or 24 hours. To prepare para formaldehyde or PFA in sucrose at 350 milliliters of PBS into a beaker and heat it to between 20 and 30 degrees Celsius at 60 grams of sucrose and allow it to dissolve before dissolving 60 grams of PFA in the solution. Then slowly add three to seven milliliters of two normal sodium hydroxide until a clear solution is obtained. After using HCL to adjust the pH to 7.4, use PBS to bring the volume to 500 milliliters.Then, with Whatman paper, filter the solution by gravity. Before use, add PBS to dilute the solution to 4% PFA and 4% sucrose. At the desired time after HLF treatment, use PBS to wash the cells twice for one minute each, and use the diluted PFA sucrose solution to fix the cells at room temperature for 20 minutes

preparing-cells-to-analyze-intracellular-uptake-activity-an-antiviral

Research

연구

JoVE Encyclopedia of Experiments

Microbiology

미생물학

Viral Growth and Techniques

바이러스 증식과 기법

Virus Detection & Imaging

바이러스 검출 및 영상화

항바이러스제의 세포 내 흡수 및 활성을 분석하기 위한 세포 준비

114 조회수. 먼저 다웰 플레이트의 커버슬립에 인간 간종 세포를 배양합니다.이 세포들은 세포질에 C형 간염 바이러스 게놈을 포함하고 있어, 바이러스 게놈 복제에 필수적인 바이러스 단백질을 생성합니다.대조 웰에서는 세포를 배양물에만 유지하고, 테스트 웰에서는 항바이러스제인 락토페린을 첨가합니다.배양판을 인큐베이팅하여 락토페린이 엔도사이토시스를 통해...

Abstract

Protocol

<table class="ck-table-resized"><tbody><tr><td>Dulbecco&#x2019;s Modified Eagle Medium (DMEM)</td><td>Wisent</td><td>319-005-CL</td><td>&#xA0;</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>BioShop</td><td>PAR070.1</td><td>Flammable solid, skin irritant, lungs and eyes</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Wisent</td><td>311-425-CL</td><td>Without calcium & magnesium ions</td></tr><tr><td>Human lactoferrin (hLF)</td><td>Sigma</td><td>L0520</td><td>&#xA0;</td></tr></tbody></table>

Preparing Cells to Analyze Intracellular Uptake and Activity of an Antiviral Agent

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재료

태그

이름	회사	카탈로그 번호	댓글
Dulbecco’s Modified Eagle Medium (DMEM)	Wisent	319-005-CL
Paraformaldehyde (PFA)	BioShop	PAR070.1	Flammable solid, skin irritant, lungs and eyes
Phosphate-buffered saline (PBS)	Wisent	311-425-CL	Without calcium & magnesium ions
Human lactoferrin (hLF)	Sigma	L0520