the pLVX-ZsGreen1 vector was assayed for p24 content using the Lenti-X p24 Titration Kit. HT1080 cells were infected with supernatant dilutions containing the indicated amounts of p24. After 48 hours, transduced cells were counted and mean fluorescence intensity was determined using flow cytometry...
The titration of the vector was carried out, using ELISA, flowcytometry, and fluorescent microscopy. Finally, transduction of HEK-293T, CHO, HepG2, MCF-7, MEFs and Jurkat cell lines was carried out with indicated cell numbers and multiplicities of infections of the vector in the presence of...
10-fold serial dilutions of a high-titer lentiviral supernatant (high, medium, and low) were concentrated from a volume of 10 ml down to 100 µl using the Lenti-X Concentrator Reagent. Titrations were performed using HT1080 cells and ...
Vector production and titration The production of HR’SIN-cPPT-SEW-eGFP-W (SIN’LTR) and its native LTR equivalent (pHV) LV was carried out as previously described [68]. These viruses have previously been used in both cell and animal assays [8,69,70,71]. The plasmid carrying eGFP flank...
Part 1:Transfection of HEK 293Cells To Produce Lentiviruses *24hours before transfection,plate 2.5X 106cells in a 10cm dish for a confluency of 50-70%the next day.Part 2:Lentiviral Harvest Part 3:Lentiviral Concentration via Ultracentrifugation Part 4:Lentiviral Titration Using Flow Cytometry Page...
analyzed by fluorescence microscopy (Panel A). Viral titers obtained for the indicated range of transfer vector input amounts were measured by qRT-PCR using theLenti-X qRT-PCR Titration Kit(Panel B). As shown, usable viral titers were obtained across a wide range of transfer...
GMP-compliant LV-induced DCs for melanoma immunotherapy BS Sundarasetty et al Titration of the vector and analyses of vector copy numbers in monocytes by RT-q-PCR For virus titration, 293 T cells were transduced and genomic DNA was extracted from using the QiaAmp DNA blood mini kit (Qiagen...
For lentivirus vectors encoding fluorescent proteins under the control of promoters functional in these cells, titration can be performed using the limiting dilution method or a flow cytometry-based method. For lentivirus vectors lacking a fluorescent marker, or for those carrying promoters that may ...
Titration of integrase defective LV-G2α2pp65 by p24 analyses Physical titers of the vectors produced (research grade and GMP grade) were determined by quantifying the p24 HIV-I core protein by ELISA (QuickTiter HIV Lentivirus Quantitation Kit, BioCat, Heidelberg, Germany). Titration of the vect...
(fluorescent)expressionwithaflowcytometer. Protocol Part1:TransfectionofHEK293CellsToProduceLentiviruses *24hoursbeforetransfection,plate2.5X10 6 cellsina10cmdishforaconfluencyof50-70%thenextday. Part2:LentiviralHarvest Part3:LentiviralConcentrationviaUltracentrifugation Part4:LentiviralTitrationUsingFlowCytometry ...