QuickTiter™ Lentivirus Quantitation Kit
Contents
1. Viral sample volume mL Virus Titer VP mL Amount of lentiviral RNA ng X 4 4 x 108 VP ng Viral sample volume mL Examples of GFP lentivirus Titer Quantitation Method 293T cells were transfected with GFP lentiviral expression construct and packaging plasmid mix Medium containing pseudotyped lentivirus was harvested and filtered after 48 hr The supernatant was then spun at 50 000 g for 1 hr to concentrate 20 fold The concentrated lentiviral supernatant titer was determined as described in assay instructions Lentiviral Supernatant 500 uL was used Average Net RFU 96 11 85 RFU or 152 ng of viral RNA Virus Titer VP mL 152 ng X 4 4 x 10 VP ng 1 3 X 10 VP mL 0 5 mL 7 AN _ CELL BIOLABS INC LA a Note The calculated result is the lentivirus physical titer and it is NOT the infectious titer TU mL The relatively large difference between the infectious titer and physical titer Viral Particles or RNA Molecules mL is derived from the large number of defective particles generated during the production process When the infectious titer is determined the results vary among different target cell lines or transduction methods References 1 Naldini L U Blomer P Gallay D Ory R Mulligan F H Gage I M Verma and D Trono 1996 Science 272 263 267 Verma I M and N Somia 1997 Nature 389 239 242 Kafri T U Blomer D A Peterson F H Gage and I M Verma 1997 Nat Genet2. Product Manual QuickTiter Lentivirus Quantitation Kit Catalog Number VPK 112 20 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Lentivirus vector based on the human immunodeficiency virus 1 HIV 1 has become a promising vector for gene transfer studies The advantageous feature of lentivirus vector is the ability of gene transfer and integration into dividing and non dividing cells The pseudotyped envelope with vesicular stomatitis virus envelope G VSV G protein broadens the target cell range Lentiviral vectors have been shown to deliver genes to neurons lymphocytes and macrophages cell types that previous retrovirus vectors could not be used Lentiviral vectors have also proven to be effective in transducing brain liver muscle and retina in vivo without toxicity or immune responses Recently the lentivirus system is widely used to integrate siRNA efficiently in a wide variety of cell lines and primary cells both in vitro and in vivo Lentivirus particles are produced from 293T cells through transient transfection of 3 or 4 plasmids that encodes for the components of the virion Viral medium containing viral particles produced by packaging cells within 48 72 hr can be harvested To ensure that pseudoviral medium is viable and to control the number of copies of integrated viral constructs per target cell the viral titer needs
3. 17 314 317 Beyer W R M Westphal W Ostertag and D von Laer 2002 J Virol 76 1488 1495 Recent Product Citations 1 2 10 11 12 Lambert M P et al 2015 Intramedullary megakaryocytes internalize released platelet factor 4 PF4 and store it in alpha granules J Thromb Haemost doi 10 1111 jth 13069 Kim H Suk et al 2015 APE1 the DNA base excision repair protein regulates the removal of platinum adducts in sensory neuronal cultures by NER Mutat Res Fund Mol M doi 10 1016 j mrfmmm 2015 06 010 Yadavilli S et al 2015 The emerging role of NG2 in pediatric diffuse intrinsic pontine glioma Oncotarget 6 12141 12155 Ahmad M A et al 2015 Label free capacitance based identification of viruses Sci Rep 5 9809 Tang X et al 2015 The advantages of PD1 activating chimeric receptor PD1 ACR engineered lymphocytes for PDL1 cancer therapy Am J Transl Res 7 460 473 Giraldo D M et al 2015 Impact of in vitro costimulation with TLR2 TLR4 and TLR9 agonists and HIV 1 on antigen presenting cell activation Intervirology 58 122 129 Shin H S et al 2015 Crosstalk among IL 23 and DNAX activating protein of 12 kDa dependent pathways promotes osteoclastogenesis J Immunol 194 316 324 Al Ahmad M et al 2014 Virus detection and quantification using electrical parameters Sci Rep 4 6831 Fan X et al 2014 Endometrial VEGF induces placental sFLT1 and leads to pregn
4. and use tube 9 as a blank Transfer 5 uL of each dilution including blank to a microtiter plate suitable for fluorometer Add 95 uL of 1X CyQuant GR Dye to each of the wells containing the 5 uL sample Read the plate with a fluorescence plate reader using a 480 520 nm filter set Pseudovirus Production The following procedure is suggested for a 10cm dish and may be optimized to suit individual needs Please refer to the user manual when the lentivirus expression systems from Invitrogen or System Biosciences is used 1 Use HEK 293T cells that have been passaged 2 3 times prior to transfection Culture these cells until the monolayer is 70 80 confluent Replace the cell culture media with new growth media 10 mL per 10 cm dish 3 Transfect cells with packaging plasmid mix and your expression construct When use Lipofectamine please refer to Invitrogen s Lipofectamine reagent manual p p g p g After 48 hrs harvest all 10 mL medium in a 15 mL conical tube and centrifuge for 5 min at 3000 rpm to pellet the cell debris Filter the supernatant through a 0 45 um low protein binding filter To concentrate the viral supernatant spin at 50 000 g for 1 hr and resuspend the viral pellet in culture medium The concentrated viral supernatant can be immediately tittered or stored at 80 C Note Freezing and thawing may result in 2 3 fold loss of viral titer after each cycle Assay Protocol 1 Add viral sample 1 to 500
5. fluorometer Add 95 uL of freshly prepared 1X CyQuant GR Dye to well s containing the 5 uL supernatant Read the plate with a fluorescence plate reader using a 480 520 nm filter set 8 Calculate lentivirus virus titer based on the standard curve Example of Results The following figures demonstrate typical quantitation results One should use the data below for reference only This data should not be used to interpret actual results 6 AN CELL BIOLABS INC N ZA a D oa o o o o o N S RFU 520 nm E N o gt D L a o oO o 400 800 Lentiviral RNA ng Lentiviral RNA ng Figure 1 Lentivirus RNA Standard Curve The QuickTiter Lentivirus RNA Standard was diluted as described in the above instructions Fluorescence measurement was performed on SpectraMax Gemini XS Fluorometer Molecular Devices with a 485 538 nm filter set and 530 nm cutoff Calculation of Lentivirus Titer VP mL 1 Determine Viral RNA amount 1 Calculate Net RFU Relative Fluorescence Unit Net RFU RFU viral sample RFU negative control corresponding to viral sample 2 Use the standard curve to determine the viral RNA amount of each unknown sample 2 Calculate Viral Titer The average genome size of lentivirus is 8 kbp therefore 1 ng lentiviral RNA 1x10 g 8 000 bp x 660 g bp X 6 x 10 1 1 x 10 VP Virus Titer VP mL Amount of lentiviral RNA ng X 1 1 x 10 VP X 20 uL 5 uL
6. its components or any materials made using the product or its components in any activity to generate revenue which may include but is not limited to use of the product or its components i in manufacturing 11 to provide a service information or data in return for payment iii for therapeutic diagnostic or prophylactic purposes or iv for resale regardless of whether they are resold for use in research For information on purchasing a license to this product for purposes other than as described above contact Life Technologies Corporation 5791 Van Allen Way Carlsbad CA 92008 USA or outlicensing lifetech com Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE CELL BIOLABS sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of CELL BIOLABS to repair or replace the products In no event shall CELL BIOLABS be liable for any proximate incidental or consequential damages in connection with the products Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbiolabs com 2004 2
7. uL to a 1 5 mL microcentrifuge tube and adjust the final volume to 1 mL with 1X PBS containing 10 mM MgClo 1 mM CaCl 5 h J CELL BIOLABS INC A SN Note A proper negative control MUST be included for accurate quantitation For purified viral sample viral storage buffer is suggested For unpurified viral supernatant use the same volume of untransfected or mock transfected 293T culture medium supernatant 2 Add 10 ul of QuickTiter Solution A to the assay tube and mix by inverting the tube several times Incubate at 37 C for 30 minutes 3 Mix the QuickTiter Lentivirus Capture Solution by vortexing for 10 seconds Quickly transfer 40 uL of the bead capture solution to the assay tube containing the viral sample Incubate at room temperature for 10 min on an orbital shaker 4 Spin down the beads at 2000X g for 30 seconds Discard the supernatant and wash the beads with 750 uL of 1X QuickTiter Solution B Mix by inverting the tube several times spin down the beads and discard the supernatant 5 Repeat the wash step once and aspirate the final wash To remove the last bit of liquid centrifuge the tube again at 2000X g for 30 seconds and remove remaining supernatant with a small bore pipette tip to avoid aspirating the beads 6 Add 20 uL of 1X QuickTiter Solution C mix with the beads by vortexing for 10 seconds spin down the beads at 12000g for 30 seconds 7 Transfer 5 uL supernatant to a microtiter plate suitable for
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9. Plate Reader Storage Store all kit components at 4 C until their expiration dates Safety Considerations Remember that you will be working with samples containing infectious virus Follow the recommended NIH guidelines for all materials containing BSL 2 organisms AN CELL BIOLABS INC JZ i Preparation of Reagents 1X QuickTiter Solution B Prepare a 1X QuickTiter Solution B by diluting the provided 10X stock 1 10 in deionized water Store the diluted solution at room temperature 1X QuickTiter Solution C Prepare a 1X QuickTiter Solution C by diluting the provided 2X stock 1 2 in deionized water Store the diluted solution at room temperature 1X CyQuant GR Dye Estimate the amount of 1X CyQuant GR Dye needed based on the number of assays including lentivirus RNA standard samples Immediately before use prepare a 1X CyQuant GR Dye by diluting the provided 400X stock 1 400 in 1X TE For best results the diluted solution should be used with 2 hrs of its preparation Preparation of Standard Curve 1 To create lentivirus RNA standards from 200 ug mL 100 ug mL 50 pg mL 25 ug mL 0 ug mL 1 2 serial dilution label nine microcentrifuge tubes 1 to 9 Add 20 uL of 1X QuickTiter Solution C to tube 2 to 9 transfer 20 uL of 200 ng mL QuickTiter Lentivirus RNA Standard to tube 1 and 2 Mix tube 2 well transfer 20 uL of the mixture 100 g mL to the next tube Repeat the steps through tube 8
10. ancy complications J Clin Invest 124 4941 4952 Zhao S L et al 2014 Mesenchymal stem cells with overexpression of midkine enhance cell survival and attenuate cardiac dysfunction in a rat model of myocardial infarction Stem Cell Res Ther 5 37 Rossello R A et al 2013 Mammalian genes induce partially reprogrammed pluripotent stem cells in non mammalian vertebrate and invertebrate species eLife Sci 2 e00036 Fan X et al 2012 Transient inducible placenta specific gene expression in mice Endocrinology 153 5637 5644 8 CELL BIOLABS INC A a 13 Veeraraghavalu K et al 2010 Presenilin 1 mutants impair the self renewal and differentiation of adult murine subventricular zone neuronal progenitors via cell autonomous mechanisms involving notch signaling J Neurosci 30 6903 6915 14 Niwano K et al 2008 Lentiviral vector mediated SERCA2 gene transfer protects against heart failure and left ventricular remodeling after myocardial infarction in rats Mol Ther 16 1026 1032 License Information This product is provided under an intellectual property license from Life Technologies Corporation The purchase of this product conveys to the buyer the non transferable right to use the purchased product and components of the product only in research conducted by the buyer whether the buyer is an academic or for profit entity The sale of this product is expressly conditioned on the buyer not using the product or
11. to be determined before proceeding with transduction experiments Viral titer can be determined by transduction of HT 1080 or Hela cells and followed by antibiotic selection of stable clones However it takes weeks to generate sizable stable cell colonies for counting and calculating the titer results Cell Biolabs proprietary QuickTiter Lentiviral Quantitation Kit does not involve cell infection instead it specifically measures the viral nucleic acid content of purified viruses or unpurified viral supernatant sample See Test Principle In the case of unpurified viral supernatant the kit is especially useful for determining the supernatant titer before the transduction step The kit has detection sensitivity limit of 1 X 10 VP mL which is sufficient for mid or high titer lentivirus sample The entire procedure takes about 45 to 60 minutes Each kit provides sufficient reagents to perform up 20 tests for viral samples and controls QuickTiter Lentiviral Quantitation Kit provides an efficient system for rapid quantitation of lentivirus titer for both viral supernatant and purified virus The system may be adapted to quantitation of other viral types such as retrovirus and adenovirus 2 CELL BIOLABS INC Ps Assay Principle How QuickTiter Kit Works 1 Viral Stock Protein RNA DNA Virus 2 Nuclei Acid Digestion Protein 2 Virus Capture 3 Protein Denaturation amp Viral Genome Release Dena
12. tured Proteins Viral Nuclei Acid 4 Quantitation Fluorescence 4 Denatured Proteins Viral Nuclei Acid QuickTiter Method patent pending CELL BIOLABS INC Creating Solutions for Life Science Research Related Products Oe OY oe Yeh ES LTV 100 293LTV Cell Line LTV 200 ViraDuctin Lentivirus Transduction Kit LTV 300 GFP Lentivirus Control VPK 104 ViraBind Lentivirus Purification Kit VPK 107 QuickTiter Lentivirus Titer Kit Lentivirus Associated HIV p24 VPK 108 H QuickTiter Lentivirus Quantitation Kit HIV p24 ELISA VPK 211 PAN ViraSafe Universal Lentivirus Expression System VPK 211 pSMPUW Universal Lentiviral Expression Vector Promoterless Kit Components 1 2 3 4 5 6 QuickTiter Solution A Part No 90020 One tube 200 uL QuickTiter Lentivirus Capture Solution Part No 90026 One tube 1 0 mL QuickTiter Solution B 10X Part No 90022 Two tubes 1 8 mL each QuickTiter Solution C 2X Part No 90023 Two tubes 1 5 mL each CyQuant GR Dye 400X Part No 105101 One tube 50 uL QuickTiter Lentivirus RNA Standard Part No 90027 One tube 500 uL containing 200 ug mL Lentivirus RNA Standard Materials Not Supplied NP ye N Lentiviral Sample purified virus or unpurified viral supernatant Cell Culture Centrifuge 0 45 um filter 1X PBS containing 10 mM MgCh 1 mM CaCl 1X TE 10 mM Tris pH 7 5 1 mM EDTA Fluorescence
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