Centrifuge the remaining culture at 150 x g for 5 minutes. Immunophenotyping cancer cells using flow cytometry on the bd accuri c6 subject. To carry out the biological characterization experiments, different dilutions of the brp05 supernatant were incubated with the antiflavivirus monoclonal antibody mab 4g2, and this mixture was used to infect c6 36 cells. The widely used mosquito cell line, c636, derived from. Viral growth was confirmed by titration of the passage material in other susceptible host systems. C636 and ap61 cells were grown in tubes 16x125 mm with screwcaps in 2 ml of growth medium. Dengue virus detection using whole blood for reverse. Tissues should be collected aseptically and rapidly. Research article chloroquine inhibits dengue virus type 2 replication in vero cells but not in c636 cells kleber juvenal silva farias, 1,2 paularenatalimamachado,1,2 and benedito antonio lopes da fonseca 1 departmentofinternalmedicine,schoolofmedicineofribeir aopreto,universityofs aopaulo, ribeir aopreto,sp,brazil. Before the availability of molecular approaches, our laboratory used direct c6 36 cell culture and toxorhynchites splendens amplification followed by c6 36 cell culture for dengue virus isolations.
Light microscopy of uninfected and zikvinfected c636 cell monolayers. Animal cell culture protocol aseptic technique and good cell culture practice to ensure all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi and mycoplasma and cross contamination with other cell lines. It was concluded that aaldnv4 may have been derived from the original dnv found in the c6 36 cell line and has transferred worldwide from the exchange of this cell line among laboratories. Development of standard methods for zika virus propagation, titration, and purification. Zika virus transmission to mouse ear by mosquito bite. To carry out the biological characterization experiments, different dilutions of the brp05 supernatant were incubated with the antiflavivirus monoclonal antibody mab 4g2, and this mixture was used to infect c636 cells.
Denv2 and denv4 stocks were produced by infecting lowconfluency huh7 or c636 cells one day postseeding at a multiplicity of infection moi of 0. Ten milliliters of culture fluid from dengue virus type 2 were. It is important to avoid excessive alkalinity of the medium during recovery of the cells. Jul 20, 2017 light microscopy of uninfected and zikvinfected c6 36 cell monolayers. As mentioned, brp05 was first detected in the fifth passage of a denv clinical sample in cell culture. This is the tmprotocol for cell seeding cells on 3d insert scaffolds in a nontreated cell culture platedish. Resuspend cells at a concentration of 24x10 6 cells per ml in freeze medium. C6 transfection reagent glioma cells, ccl107 altogen. Quantification, propagation, detection, and storage. The optimal method for determining specific etiology of an. New reverse genetics and transfection methods to rescue. Cultivation of mosquito cell lines in serumfree media and. The susceptibility of the c636 clone of aedes albopictus monolayer cell cultures was determined with 46 prototype viruses passed through three subcultures.
Development of standard methods for zika virus propagation. What is the best protocol for reviving and subculturing c636 walbb. But the recommended temperature for culturing this cell lines is 28 oc with 5%co2. By using a dynamical model of such kind of processes, an optimizationbased technique for estimation of kinetic parameters in the model of mammalian cell culture process is developed. Structural investigation of c636 and vero cell cultures. Mosquito inoculation is the most sensitive isolation method but is impractical for. Altogen biosystems provides twocomponent highefficiency transfection reagent kits for the c6 glioma cell line. Cell culture is the process by which cells are grown under controlled conditions, generally outside their natural environment. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. Cell culture and fluorescent staining page 1 culturing and fluorescent staining of b16 melanoma cells in this lab exercise you will learn how animal cells can be grown in culture and used to analyze the cytosolic arrangement of actin filaments.
Culturing and fluorescent staining of b16 melanoma cells. Before the availability of molecular approaches, our laboratory used direct c636 cell culture and toxorhynchites splendens amplification followed by c636 cell culture for dengue virus isolations. Cell culturing, and the requisite storage of cell lines at ultralow. Subculturing adherent cells thermo fisher scientific us. Monolayer cultures of the tra284 cells were prepared by seeding 4 million cells per flask, whereas monolayers of the c6 36 and ap61 cells were prepared by seeding 1 million cells per culture tubeheld in aslant rack. Currently, polymerase chain reaction pcr is the method of choice for rapid and early virological diagnosis of dengue infections, but viral. For passaging your own cell line, we recommend that you. Flow cytometrybased assay for titrating dengue virus. Apr 30, 2009 i would like culture c636 insect cell lines. Mosquito cell line c636 shows resistance to cyt1aa6.
Comparative sensitivity of mosquito cell lines for isolation. Enquiries regarding the commercial use of a cell line are referred to the depositor of the cell line. Factors influencing dengue virus isolation by c636 cell. I am having issues with reviving c636 walbb c636 infected with wolbachia from the mosquito aedes albopictus. Culture of hek 293293t cells charles yin last updated. After axons have grown in vitro, c6 cells are added to the vicinity of axons and contact the axons. What is the best protocol for reviving and subculturing c6 36 walbb c6 36 infected with wolbachia from the.
Igarashi and then cloned and recloned by seeding single cell suspensions into petri dishes. Aedes albopictus clone c636 atcc crl1660 aedes albopic. Purification of dengue virus particles by onestep ceramic. Volumes used in this protocol are for 75 cm 2 flask.
Subculture and maintenance 1 maintain culture at a cell density between 1x105 and 3x106 cellsml. The 50year old aedes albopictus c636 cell line is a resource for the detection. Aedes albopictus c636 cells 5 were grown at 28c in l15 media. This data sheet shows the kind of rich data you can generate using the bd accuri c6 personal flow cytometer for two kinds of cancer biology studies. Cholera toxin induces malignant glioma cell differentiation. Procedure 1 sanitize the cabinet using 70% ethanol before commencing work. 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. Whole blood was analyzed by extracting rna from 100 l of each spiked wholeblood sample with denv1 at 102 to 104 pfuml, and the remaining blood was used for plasma or serum preparation. Viral isolation protocol for cell culture introduction. It could easily be integrated in others multiprotocol instant messengers.
The column was then equilibrated with 10 mm napb ph 7. Note that the vial of bhk cells contains 3 x 106 cells. Susceptibility of other cell culture systems to infection. Compatibility of symbionts with dipteran and lepidopteran cell lines. The uninfected c636 cells are regularly disposed in monolayer covering the entire culture plate a. Prepare transfection complexes by mixing 40 l of serum. Remove the vial of cells from the liquid nitrogen and thaw quickly at 37c. It is suggested that, prior to the addition of the vial contents, the culture vessel. Cell culture protocol for freezing cell lines at high cell viabilities using cryopreservation reagents such as sigma dmso.
Note that cell culture conditions vary for each cell type. To evaluate the ability of c6 36 and vero cells to produce zikv progeny, realtime rtpcr was performed on cell supernatant samples collected at different timepoints of infection. Migration of c6 cells on chick retinal axons in culture. Effect of the temperature on the efficiency of virus rescue and on the efficacy of transfection in c636 cells. Ten milliliters of culture medium from c636 cell cultures infected with secondary symbionts from a. Ideally, the cell viability should be in excess of 90% in order to achieve a good recovery after freezing. 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. Factors influencing dengue virus isolation by c6 36 cell culture and mosquito inoculation of nested pcrpositive clinical samples. Distinctly, the c6 36 monolayers that were cultivated following inoculation with zikvinfected sgs are showing adjacent cells forming syncytia with empty areas on the. C6 36 and ap61 cells were grown in tubes 16x125 mm with screwcaps in 2 ml of growth medium. Distinctly, the c636 monolayers that were cultivated following inoculation with zikvinfected sgs are showing adjacent cells forming syncytia with empty areas on the. Two or three days before dissection of mosquito tissue, grow c636 cell in a 24 well plate such that the cell reach 80% confluency at the day when the assay is conducted.
Analysis of the aedes albopictus c636 genome provides. The zikv sample used was isolated in c636 cells from brazilian patient blood in 2015 by the flavivirus reference laboratory, instituto oswaldo cruz, fiocruz human research ethic committee protocol number. Currently, polymerase chain reaction pcr is the method. Monolayer cultures of the tra284 cells were prepared by seeding 4 million cells per flask, whereas monolayers of the c636 and ap61 cells were prepared by seeding 1 million cells per culture tubeheld in aslant rack. Note that the procedure for passaging insect cells differs from that for mammalian cells on several crucial steps. Aphidsymbiotic bacteria cultured in insect cell lines. A new medium for adenovirus production sigmaaldrich. A new aura virus isolate in brazil shows segment duplication. In vitro cell culture methods using mosquito cell lines c636 and other. Insect cell lines are very sensitive to dengue virus. It could easily be integrated in others multi protocol instant messengers. Rapid cryopreservation of five mammalian and one mosquito cell.
Initiating cell culture from frozen stock the following protocol is designed to help you initiate a cell culture from a frozen stock. Oct 25, 2017 effect of the temperature on the efficiency of virus rescue and on the efficacy of transfection in c6 36 cells. C6 36 cell culturescloned aedes albopictus mosquito. The utilization of tick cell lines for the isolation of rickettsia has become an excellent tool for previously uncultivated organisms 36, 37 as well as for protein expression analysis and genetic manipulation of sfgr in an arthropodderived host cell 4, 5, 14, 26. May 29, 2018 as mentioned, brp05 was first detected in the fifth passage of a denv clinical sample in cell culture. Immunophenotyping cancer cells using flow cytometry on. At 48 hours posttransfection the cells were analyzed by qrtpcr for lamin ac gene.
Protocol 2 generation and purification of zika virus stocks zikv can be easily propagated in mammalian and mosquito cell lines e. An improved method of isolation and identification reported by tesh 15. What is the best protocol for reviving and subculturing c636 walbb c636 infected with wolbachia from the. Sample 2, culture fluid of c636 cells without virus infection. Comparative sensitivity of mosquito cell lines for. Procedure available tissues or fluids should be divided for viral isolation, electron microscopy, and for immunohistochemical examination. In contrast, cells in s phase were dramatically decreased from 36. Viral stocks were obtained by inoculating a monolayer of c636 cells in a 75cm2 tissue culture. Factors influencing dengue virus isolation by c636 cell culture. It was concluded that aaldnv4 may have been derived from the original dnv found in the c636 cell line and has transferred worldwide from the exchange of this cell line among laboratories. Research article chloroquine inhibits dengue virus type 2. Zikv rna detection from c6 36 and vero cell culture supernatants. Factors influencing dengue virus isolation by c636 cell culture and mosquito inoculation of nested pcrpositive clinical samples.
The resistant c636 cells were selected from c636 cells in vitro activated cyt1aa6 at generation 10. L of the patient sample and incubated for 1 hour hr at 28c for. The uninfected c6 36 cells are regularly disposed in monolayer covering the entire culture plate a. Denv2 and denv4 stocks were produced by infecting lowconfluency huh7 or c6 36 cells one day postseeding at a multiplicity of infection moi of 0.
Splitting can be performed by centrifuging cells at 500g for 5 minutes, decanting growth medium and rinsing in. Culture supernatant was harvested six days postinfection, cell debris pelleted at 1,300 rpm for 5 min and cleared supernatants used as virus stocks. Reagents vero cell culture monolayers or other suitably. Once we established that 5 days of infection and 1. A coculture assay to visualize and monitor interactions. Cell culture protocols thermo fisher scientific za. Cell supplemental information defining hsp70 subnetworks. Wolf, university of maryland, baltimore county general guidelines to cell and tissue culture techniques, including. The following protocol describes a general procedure for subculturing adherent mammalian cells in culture. Aedes albopictus, densovirus, c636, genetic variation. Aedes albopictus atc15 atcc ccl126 aedes albopictus l. Cell supplemental information defining hsp70 subnetworks in. May 21, 2009 migration of c6 cells on chick retinal axons in culture.
It is critical to follow this 3d cell seeding protocol. Other notes cultures from hpa culture collections and supplied by sigma are for research purposes only. Research article chloroquine inhibits dengue virus type 2 replication in vero cells but not in c636 cells. Aedes albopictus, densovirus, c6 36, genetic variation. We therefore recommend that you familiarize yourself with your cell. Cell cultures derived from larvae of aedes albopictus skuse and aedes aegypti l. Aaldnv found in c6 36 cell line than to aaednv isolated from other mosquitoes. Aug 14, 2007 in contrast, cells in s phase were dramatically decreased from 36. Susceptibility of aedes albopictus c636 cells to viral infection. The susceptibility of the c6 36 clone of aedes albopictus monolayer cell cultures was determined with 46 prototype viruses passed through three subcultures.
For more information, refer to notes on subculturing insect cells. Use of monocytederived macrophage culture increases zika. Following this protocol will allow the cell suspension to. Sample 2, culture fluid of c6 36 cells without virus infection. The present work is dedicated to nonlinear dynamic modelling and parameter estimation for a mammalian cell culture process used for mab production. Growth and maintenance of bhk cells thermo fisher scientific. Mosquitoes are anesthetized at 4c, sacrificed and surfaced sterile by soaking them in 75% ethanol for 1 minute. Here we describe propagation using the vero e6 cell line. Nineteen viruses demonstrated good growth in c636 cells. C636 cell culturescloned aedes albopictus mosquito cells.
Virus isolation in cell culture is to be preferred, however. Aaldnv found in c636 cell line than to aaednv isolated from other mosquitoes. C636 cell culturescloned aedes albopictus mosquito. Susceptibility of aedes albopictus c636 cells to viral. Mammalian cell culture process for monoclonal antibody.
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