Since then, many cell lines have been derived from human colon cancers. Characterization of side population cells isolated from the colon. Early cell culture research focused on discovering methods for culturing a diverse array of cells from many species. Remove a volume of the single cell suspension and dilute with trypan blue solution and magnetic cell. Cell fusion was performed according to the method of davidson. Remove a volume of the single cell suspension and dilute with trypan blue solution and magnetic cell sorting buffer macs buffer for counting. We selected the 120 split from a few days previous. I am working with colorectal cancer cell linesht29 and sw480 and breast cancer cell line mcf7. Sw480 cell line alvetex scaffold plates and inserts. Silencing of the htert gene by shrna inhibits colon cancer. Cell culture cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells e. In most cases, cultures at a high cell density exhaust the medium faster.
Briefly, sw480 cells were seeded as reported in section 4. Observe culture daily by eye and under an inverted microscope to ensure culture is free of contamination and culture has not reached confluence. Check the ph of the culture medium by looking at the color of the indicator, phenol red. Dislodge cells from the flask substrate with a cell scraper. Primary tumor and metastasisderived colon cancer cells differently. Take a closer look at exosomes thermo fisher scientific. Phase contrast micrographs of sw480 cells left, sw620 cells middle and 3t3 cells. Example protocols for the coculture of either the sw620 or. After 7 days, inserts were transferred to deep well petri dish holders in deep well petri dishes. Initially, this cell line was used to study different aspects of the biology of human cancers. Transfer the cell suspension into the culture vessel containing the complete growth medium and mix thoroughly by gentle rocking.
Exosomes derived from sw480 colorectal cancer cells promote. Altogen biosystems provides polymerbased transfection kits for the sw480 cell line. In vitro analysis of putative cancer stem cell populations. Note that cell culture conditions vary for each cell type. Cell culture basics handbook thermo fisher scientific.
Silencing of the htert gene by shrna inhibits colon cancer sw480 cell growth in vitroand in vivo aiqun liu1. Coculture of sw620 or sw480 with 3t3 cell line alvetex scaffold inserts page 3 of 5 5. And please pay special attention to avoid contamination. Sw480 cell line alvetex scaffold plates and inserts page 2 of 5 methods preparation for 3d cell culture on alvetex scaffold 1. Cell culture protocols university of california, san diego. Pdf optimization of sw480 colon cancer cells transfection with. Ht29p no165, sw480 passage no 178 and mcf7 p no 11. Plates were reincubated and maintained for a further 7 days by complete 3t3 media exchange after every 23 days. This protocol guide details steps on how to thaw, culture and cryopreserve human ipscs supplied by the european bank of induced pluripotent stem cells ebisc.
The ability of sw480 cancer cells to form colonies in culture plates was. The sw480 cell line was obtained from the american. Transfection protocol of adherent sw480 cells 96well plate. Quantitative proteomic analysis of 2d and 3dcultured sw480 cells treated with xav939. A confluent monolayer is an adherent cell culture dish, plate or flask in which the cells have formed a single layer over the entire surface area available for growth. This method can be applied to the use of alvetex scaffold in12 well plate format. Atcc offers custom testing services for mycoplasma detection and human cell line authentication str analysis. Initially, this cell line was used to study different aspects of. Subculturing adherent cells thermo fisher scientific in. Example protocol for the culture of the sw480 cell line on. Isolation and characterizationof exosomes from cell culture. Apoptosis induction pathway in human colorectal cancer cell. Wash cells briefly with hanks solution prior trypsin treatment note. Fusobacterium nucleatum promotes chemoresistance to.
Sw480 sw480 atcc ccl228 homo sapiens colon dukes typ. Take a closer look at exosomes vesicle transport and cell signaling the 20 nobel prize in physiology or medicine was jointly awarded to three scientists for their discovery of vesicle transport in cells, a clear indication of the importance of vesicles in physiology and their applied potential in diagnostics and therapy. Sw480 and sw620 cells were seeded at a density of 3. Simplifying methods for exosome enrichment and analysis abstract exosomes, 50150 nm sized vesicles, are secreted by all cells and found in all body fluidsinvestigated. Hct116, lovo, sw480 and colo205 colon tumor cell lines were. Realtime cell analysis was performed using the xcelligence system roche applied science, rotkreuz, swtizerland. This study aimed to identify cancer stem cells cscs in the sp of the colon cancer cell line sw480. Cell line authentication str profiling aids in the detection of misidentified, crosscontaminated, or genetically drifted cells, which invalidate research results. Sw480 and hct116 cells were procured from american type culture collection manassas, va and maintained in leibovitz medium and dulbeccos modified eagle medium, respectively, supplemented with 10% fbs and 100 uml of penicillin and streptomycin under standard culture conditions. Culture of hek 293293t cells charles yin last updated. We reported that sp cells isolated from crc cell line exhibited higher.
Take a closer look at exosomes vesicle transport and cell signaling the 20 nobel prize in physiology or medicine was jointly awarded to three scientists for their discovery of vesicle transport in cells, a clear indication of the importance of vesicles in physiology and their applied potential in. Confluency confluency is a marker for when to subculture your cells. The human colon adenocarcinoma cell line ht29 was isolated from a primary tumor of a 44 years old caucasian female in 1964 by fogh and trempe. This is the complete description of the cell line that you selected. Isolation and characterization of a highly malignant. The goal of this study was to understand if exosomes derived from highmetastatic cells may influence the behavior of less aggressive cancer cells. Isolation and characterization of a highly malignant variant of the sw480 human colon cancer cell line naohiro. The different cell lineage, tumor histotype, method of isolation. When subculturing cells 6075% confluency, discard old media from flask. Invitrogen cellsensor leftcf bla sw480 cellbased assay protocol page 3 of for technical support for this or other invitrogen drug discovery solutions products, dial 760 603 7200, extension 40266 4. Fusobacterium nucleatum promotes chemoresistance to colorectal cancer by modulating autophagy.
We found that the human colon cancer cell line sw480 consists of. Side population sp cells may play a crucial role in tumorigenesis and the recurrence of cancer. Transfection protocol of adherent sw480 cells 96well plate 1. Transfection and selection data of sw480 cells by various antibiotics in differing media. Add a volume of equilibrated, fresh growth medium the volume of the culture volume. Sw480 transfection kit colon carcinoma, ccl228 altogen. If the medium goes from red through orange to yellow, change the medium. Quantitative proteomic analysis of 2d and 3d cultured. Cell culture sw480, sw620, hct116 cell lines were obtained from china infrastruture of cell line resources and. Examine the cell cultures after 24 hours and subculture as needed. Design and equipment for the cell culture laboratorycontents. The consequences of deviating from the culture conditions required for a particular cell type can range from the expression of aberrant phenotypes to a complete failure of the cell culture. Co culture of sw620 or sw480 with 3t3 cell line alvetex scaffold inserts page 2 of 5 methods preparation for 3d cell culture on alvetex scaffold 1. Silibinin suppresses growth of human colorectal carcinoma.
The human hepatocellular cancer hepg2 cells jcrb1054 were purchased from the health science research resources bank osaka, japan. The human colon cancer cell lines sw480, lovo and hct116 contain. After 7 days, inserts were transferred to deep well petri dish holders in. Establishment and characterization of a new cell line derived from. Induced pluripotent stem cell culture protocols sigmaaldrich. In most cases, cultures at a high cell density exhaust the medium.
Isolation and characterization of a highly malignant variant. The goal of this study was to understand if exosomes derived from highmetastatic cells may influence the behavior of less aggressive cancer cells and the properties of the endothelium. Silibinin suppresses growth of human colorectal carcinoma sw480 cells in culture and xenograft through downregulation of catenindependent signaling 1. Note 2 processing flask cultures some atcc cell, are shipped as growing cultures in culture vessels. The sw480 cell line was obtained from the american type culture collection. After 48 h, cell culture media were collected, and exosomes were isolated by a multistep centrifugation protocol. The odd thing about these sw480 is that apart from the regular epithelial morphology that ive observed, there seem to be odd cell morphologies forming in the culture as well as shown in the attached picture. Example protocol for the culture of the sw480 cell. Exosomes from metastatic cancer cells transfer amoeboid. Pdf silibinin suppresses growth of human colorectal. We investigated the effects of xav939 on the cell growth of 2d and 3dcultured human crc sw480 cells. Pdf 9 mb download pdf 9 mb download figures ppt add to online library powered by mendeley. The human colon cancer cell lines sw480, lovo and hct116 contain 1.
Induction of apoptosis of sw480 human colon cancer cells by. Gently pipette up and down to create a single cell suspension using a 10ml pipette. This cell line is widely used in biomedical research related to the search for treatments for colon cancer. Upon heavy growth of tumor cells, the adherent colonies were scraped off under a stereomicroscope for further culture. Download altogen biosystems sw 480 transfection protocol. Volumes used in this protocol are for 75 cm 2 flask. To identify the cause of sw480 cell death, apoptosis induction and the permeability of cell membranes was assessed using the apopercentage kit and protocol. Pdf validation of a model of colon cancer progression. For more than 25 years, sigma and ecacc have both been part of and contributed to that advancement.
Secure a 6cm plate of hek293t cells from the stock incubator. Characterization of side population cells isolated from the. Sw480 cell line was established from the primary tumor whereas. Human induced pluripotent stem cell ipsc lines are different from any other established cell line. Click on each item for the direct links to order from available suppliers. Cell culture protocols thermo fisher scientific kr. Gjic between hmec was measured by means of gapfrap method 18.
Sw480 cells were routinely maintained in t75 flasks. We investigated the effects of xav939 on the cell growth of 2d. Assay for phenotype or target gene expression 48 72 hours after transfection 1. Ncipbcfccl228 sw480 colon adenocarcinoma atccccl228 february 27, 2012. After this time, the sp sorting method was applied to the two groups to. Barley and oat phenolic extracts at a concentration of 1500 gml were. Ive recently started growing sw480 ordered from atcc about slightly less than a month ago. Exosomes derived from sw480 colorectal cancer cells.
Many types of cell lines and tissues have demonstrated the presence of sp cells, including colon cancer cell lines. Recommended transfection protocols for 24well plate. For passaging your own cell line, we recommend that you closely follow the instructions provided with each product you are using in your experiments. Flow cytometry of ectreated sw480 cells using a doublestaining method with. A cell line established from a lymph node metastasis taken from the same patient one year later is available see atcc ccl227. Image shows cells at low left and high right confluency. Example protocols for the coculture of either the sw620. Cellsensor leftcfbla sw480 cellbased assay protocol. Observe culture daily by eye and under an inverted microscope to ensure culture is free of. Invitrogen cellsensor leftcf bla sw480 cell based assay protocol page 3 of for technical support for this or other invitrogen drug discovery solutions products, dial 760 603 7200, extension 40266 4.
After selection with 1 mgml of g418 gibco laboratories to obtain neo clones or. We would like to show you a description here but the site wont allow us. The human colorectal cancer sw480 cells ccl228 were purchased from the american type culture collection atcc, manassas, va, usa. Half maximal inhibitory concentration, or ic 50, is a measurement representing the halfway point in which a compound of interest produces complete inhibition of a biological or biochemical function. Phase contrast micrograph of sw480 cells grown in conventional 2d culture plates. I am a masters student performing cell culture for the first time. Cell culture and cell viability assay sw480 and hct116 cells were procured from american type culture collection manassas, va and maintained in leibovitz medium and dulbeccos modified eagle medium, respectively, supplemented with 10% fbs and 100 uml of penicillin and streptomycin under standard culture conditions.
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