Para la obtencion de ADN viral, monocapas de celulas MDBK
fueron crecidas en botellas de cultivo celular de 150 [cm.sup.2] de superficie (T150) e infectadas con las cepas de referencia y los aislamientos de campo.
Total protein of SK-6 cells infected with Linda virus and MDBK
cells infected with BVDV-1 (strain NADL) was probed with the pestivirus E2-specific antibody 6A5.
Different concentrations of P34 (1.0 to 5.5[micro]g[mL.sup.-1]) were added onto confluent monolayers of MDBK
cells and the cellular viability was assessed after 72h of incubation by the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) method and neutral red assay as described elsewhere (MOSMANN, 1983; BORENFREUND & PUERNER, 1985).
For evaluation of antiviral activity of EP1 and EP2 diverse cells (MDBK
, MDCK and CRFK) were infected with the viruses (25 pl/well of viral dilution), incubated for 2h and the inoculum was removed.
Activity against normal cells including Vero and MDBK
cells were also checked and found that MKA 17, KML 2 and SSA13 all three showed impressive IC50 values (>100 ug/ml, 56.0 ug/ml and 64.8 ug/ml respectively) against Vero cells and IC50 values are >100 ug/ml for the strains KMF 2, KME 1 and LSA 14 against MDBK
cells (Table II).
Briefly, nuclear lysates isolated from MDBK
cells were sonicated, and cross-linked proteins were immunoprecipitated by incubation with antibody against Sp1 (Santa Cruz Biotechology Inc., Dallas, Texas, USA) as well as normal IgGs (Cell Signaling Technology, USA), respectively.
The sera-virus mixture was incubated at 37[grados]C for 24 hours in 5% C[O.sub.2] and then a suspension of 50ul of MDBK
cells at a concentration of 2 x [10.sup.4] cells/well was added.
cells were inoculated with SV56/90 strain at a multiplicity of infection of 0.1.
Monolayer cultures of Madin-Darby bovine kidney (MDBK
) cells (National Bank for Industrial Microorganisms and Cell Cultures, Sofia, Bulgaria) were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (Gibco BRL, Paisley, Scotland, UK), supplemented with 10 mM HEPES buffer (2-[4-(2-hydroxyethyl)-l-piperazinyl]-ethanesulfonic acid) (Merck, Germany) and antibiotics (penicillin 100 IU/ml, streptomycin 100 [micro]g/ml) in C[O.sub.2] incubator (HERAcell[R] 150, Heraeus, Germany), at 37[degrees]C/5% C[O.sub.2].
A number of cell lines of mammalian origin i.e., BHK-21, MDCK, MDBK
, HeLa, and Vero are employed in the animal virology laboratories.
Our results from qPCR analysis (Figure 1(a)) indicated that PPARG expression is very high in all adipose tissues, followed by rumen, Madin-Darby Bovine Kidney cell line (MDBK
), and placenta with moderate-to-low mRNA expression in small intestine, beef cattle longissimus muscle, hoof corium, lung, and mammary gland.
Terminado el tiempo de incubacion, una suspension de 4 x [10.sup.6] celulas MDBK
(Madin Darby Bovine Kidney) en MEM fue adicionada en cada una de las cavidades de la placa.