QuickTiter™ Adenovirus Titer Immunoassay Kit
Contents
1. Product Manual QuickTiter Adenovirus Titer Immunoassay Kit Trial Size Catalog Number VPK 109 T 20 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Recombinant adenoviruses have tremendous potential in both research and therapeutic applications There are numerous advantages they provide when introducing genetic material into host cells The permissive host cell range is very wide The virus has been used to infect many mammalian cell types both replicative and non replicative for high expression of the recombinant protein Recombinant adenoviruses are especially useful for gene transfer and protein expression in cell lines that have low transfection efficiency with liposome After entering cells the virus remains epichromosomal i e does not integrate into the host chromosome so does not activate or inactivate host genes Recently recombinant adenoviruses have been used to deliver RNAi into cells HEK 293 cells or their variants are used as host cells for viral amplification Recombinant adenoviruses can be grown at high titer 10 VP viral particles mL which can be concentrated up to 10P VP mL and purified by Cell Biolabs ViraBind Adenoviral Purification Kit or traditional CsCl ultracentrifugation A particular challenge in the delivery of a gene by a viral vector is the accurate measurement of virus titer Traditional2. Titer Related Products jo e c DN OU d ee ce VPK 106 QuickTiterTM Adenovirus Quantitation Kit VPK 110 QuickTiterTM Adenovirus Titer ELISA Kit VPK 111 Rapid RCA Assay Kit VPK 252 RAPAd CMV Adenoviral Expression System AD 100 293AD Cell Line VPK 099 ViraBind Adenovirus Miniprep Kit VPK 100 ViraBind Adenovirus Purification Kit AD 200 ViraDuctin Adenovirus Transduction Reagent VPK 112 QuickTiter Lentivirus Quantitation Kit 10 VPK 120 QuickTiter Retrovirus Quantitation Kit Kit Components pt de ee c Anti Hexon Antibody 1000X Part No 10901 T One 6 uL tube Secondary Antibody HRP Conjugate 1000X Part No 10902 T One 10 uL tube DAB Substrate 25X Part No 10903 T One 0 5 mL tube Diluent 10X Part No 10905 One 1 5 mL tube Ad gal Positive Control Part No 10904 One tube 50 uL at 1 0 x 10 ifu mL cm E Creating Solutions fo JN CELL BIOLABS INC Research Materials Not Supplied Recombinant adenovirus of interest HEK 293 cells and cell culture growth medium Methanol 1 BSA PBS H202 Light Microscope optional p Galactosidase Staining Kit Cat AKR 100 Arp A de v qp r5 Storage Upon receipt store the Ad D gal Positive Control at 80 C Store all other 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 guideline
3. al 2009 Systemic administration of a conditionally replicating adenovirus targeted to angiogenesis reduced lung metastases burden in cotton rats Clin Cancer Res 15 1664 1673 Troidl K et al 2009 Actin binding Rho activating protein Abra is essential for fluid shear stress induced arteriogenesis Arterioscler Thromb Vasc Biol 10 1161 ATVBAHA 109 195305 Soesanto Y et al 2008 Regulation of Akt signaling by OGIcNAc by euglycemia Am J Physiol Endocrinol Metab 295 E974 E980 Black S A et al 2008 TGFBI stimulates connective tissue growth factor CCN2 CTGF expression in human gingival fibroblasts through a RhoA independent Rac1 Cdc42 dependent mechanism statins with forskolin block TGFB1 induced CCN2 CTGEF expression J Biol Chem 283 10835 10847 Akai S et al 2007 Knock Down of gamma glutamylcysteine synthetase in rat causes acetaminophen induced hepatotoxicity J Biol Chem 282 23996 24003 Saavedra L et al 2007 Internalization of beta amyloid peptide by primary neurons in the absence of apoE J Biol Chem 282 35722 35732 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 p
4. staining results 9 Aspirate DAB wash twice with 1X PBS and add 1 mL of 1X PBS to each well 10 Count positive stained cells brown for at least five separate fields per well using a light microscope and 10X objective 11 Calculate the average number of positive cells per well and viral titer infectious units mL Example of Results The following figures demonstrate typical titration results One should use the data below for reference only This data should not be used to interpret actual results 10 10 a M i Figure 2 Ad gal Titration Different dilutions of the Ad f gal positive control were used to infect HEK 293 cells for 48 hrs Anti Hexon immunostaining was performed as described in Assay Instructions and X gal staining is done by using B Galactosidase Staining Kit Cat AKR 100 10 5 Anti Hexon 6 CELL BIOLABS INC Creating Solutions for Life Science Research Calculation of Adenovirus Titer Infectious Units mL 1 Calculate the average number of positive cells per field Ideally choose a dilution with 5 50 positive cells field and count at least five fields 2 Determine the number of fields per well For most microscopes a standard 10X objective lens with 10X eyepiece lens has a field diameter D of 1 8 mm then Area per field 3 14 x D 2 3 14 x 0 9 2 54 mm For 24 well plate area of a well standard 24 well plate is 2 0 cm therefore Fields well 2 0 cm 2 54 mm 2
5. 0 cm 2 54 x 10 cm 79 For 12 well plate area of a well standard 12 well plate is 3 8 cm therefore Fields well 3 8 cm 2 54 mm 3 8 cm 2 54 x 10 cm 150 Note If you are not sure about the field diameter of the 10X objective lens you are using or you are using objective lenses other than 10X the field diameter can be determined by aligning with the grids of hemacytometer Figure 2 or referring to Table 2 TM m es HER zi EH L7 pp Wl i LLCCLLLLLILLL LL 1mm f Figure 2 Hemacytometer Grid Dimensions Objective Lenses Eyepiece Lenses 10X Fields Well Total Field Field Area 12 well 24 well Magnification Diameter mm Plate Plate 4X 40X 5mm 19 6 19 10 10X 100X 1 8 mm 2 54 150 79 20X 200X 0 9 mm 0 64 594 313 Table 2 Field sizes of objective lenses CELL BIOLABS INC Ps UR Calculate viral titer Infectious Units or ifu mL Tests in 24 well Viral Titer ifu mL average positive cells field x 79 fields well x dilution factor 0 1 mL Tests in 12 well Viral Titer ifu mL average positive cells field x 150 fields well x dilution factor 0 1 mL Calculation Example A serial of 10 fold dilutions of the provided Ad f gal Positive Control was made and its titer was determined in a 24 well plate as described in assay instruction Ten fields were counted and the average positive cells field is 12 for 1 10 dilution und
6. er a standard 10X objective therefore Viral Titer ifu mL average positive cells field x 79 fields well x dilution factor 0 1 mL Viral Titer ifu mL 12 field x 79 fields well x 10 0 95 x 10 ifu mL 0 1 mL References 1 Bewig B and W E Schmidt 2000 Accelerated titering of adenoviruses BioTechniques 28 870 873 Recent Product Citations l 2 3 Yang Y et al 2015 RGD modifided oncolytic adenovirus exhibited potent cytotoxic effect on CAR negative bladder cancer initiating cells Cell Death Dis 6 e1760 Nakao S et al 2015 Stimulus dependent regulation of nuclear Ca 2 signaling in cardiomyocytes a role of neuronal calcium sensor 1 PLoS One 10 e0125050 Patsouris D et al 2014 Insulin resistance is associated with MCP1 mediated macrophage accumulation in skeletal muscle in mice and humans PLoS One 9 e110653 Xiong Y et al 2014 Long term exposure to L arginine accelerates endothelial cell senescence through arginase II and S6K1 signaling Aging Albany NY 6 369 Barrett A et al 2014 A crucial role for DOK1 in PDGF BB stimulated glioma cell invasion through p130Cas and Rapl signalling J Cell Sci 127 2647 2658 Wilkins H et al 2013 Mitochondrial glutathione transport is a key determinant of neuronal susceptibility to oxidative and nitrosative stress J Biol Chem 288 5091 5101 Scallan C et al 2013 An adenovirus based vaccine with a double st
7. f culture medium 2 Label six sterile tubes 1 to 6 and add 900 uL of culture medium to each tube Add 100 uL of 1 100 diluted viral sample to tube 1 mix tube 1 well Transfer 100 uL of the mixture 1 1000 dilution to the next tube Repeat the steps until tube 5 and use tube 6 as a blank Note An Ad f gal Positive Control is provided as an assay control you may include 1 10 and 1 10 dilutions of this stock in your assay Ad f gal expression can also be visualized by X gal staining Assay Protocol The instructions below are suggested for assays performed in 24 well plate Use twice as much the amount of cells and reagents for assays performed in 12 well plate I Virus Infection 1 3 Harvest HEK 293 cells and resuspend cells in culture medium at 2 5 x 10 cells mL Seed 1 mL in each well of a 24 well plate and incubate at 37 C 5 CO for 1 hr Note Adenovirus titer assay is critically dependent of the firm attachment of cells If the cells look thin and easy to come off during immunostaining steps you won t get consistent result Only use low passage 293 cells with flattened morphology or 293AD Cat AD 100 a selected 293 cell line for plasmid transfection adenovirus amplification and tittering To improve cell adhesion you can also precoat plate with polylysine or extracellular matrix Prepare a 10 fold serial dilution of your viral sample in culture medium Dropwise add 100 uL of diluted viral sample to eac
8. h well of the 24 well assay plate note a negative control should be performed simultaneously To ensure accuracy perform each sample in duplicate Incubate infected cells at 37 C 5 CO for 2 days II Immunostaining 1 Slowly remove medium from the wells by tilting the plate and aspirating from the edge then fix infected 293 cells by gently adding 0 5 ml of cold methanol down the side of each well of the 24 well assay plate taking care not to dislodge the cells Incubate 20 minutes at 20 C Gently wash the fixed cells three times with 1X PBS five minutes each wash 3 Block for 1 hr with 1 BSA in PBS at room temperature on an orbital shaker Add 0 25 mL of diluted 1X anti Hexon antibody solution to each well and incubate for 1 hr at room temperature on an orbital shaker Gently wash the fixed cells three times with 1X PBS five minutes each wash Add 0 25 mL of diluted 1X Secondary antibody solution HRP conjugated to each well and incubate for 1 hr at room temperature on an orbital shaker Gently wash the fixed cells five times with 1X PBS five minutes each wash o gt CELL BIOLABS INC 8 Add 0 25 mL of freshly diluted 1X DAB working solution to each well and incubate for 10 minutes at room temperature on an orbital shaker Note Adenovirus infected cells should show dark brown staining within 5 minutes During incubation excess DAB starts to form light precipitates in solution and this will not affect the
9. ly infectivity particles are measured in culture by a plaque forming unit assay PFU that scores the number of viral plaques as a function of dilution These methods are time consuming 10 days require a long infection period and suffer from a high degree of inter assay variability and are affected by virus cell interactions Cell Biolabs QuickTiter Adenovirus Titer Immunoassay Kit utilizes an antibody against adenovirus hexon proteins to visualize infected cells by immunocytochemistry staining The hexon proteins are the largest and most abundant of the structural proteins in the adenovirus capsid and they are distributed symmetrically to form capsid facets Cell Biolabs QuickTiter Adenovirus Titer Immunoassay Kit provides a quick and complete system to functionally titer virus infectivity This Trial Size kit provides sufficient reagents for up to 20 titrations in a 24 well plate In contrast to the 10 day infection of a classical plaque assay this kit only requires a 2 day infection The kit antibody against hexon protein recognizes all 41 serotypes of adenovirus by immunocytochemistry and can be used with any adenovirus system as long as the virus is able to amplify in HEK 293 cells CELL BIOLABS INC Assay Principle Seed 293 cells in 24 or 12 well plate for 1 hr Prepare Adenovirus Serial Dilutions and Infect 293 cells for 48 hrs Anti Hexon Immunocytochemistry Staining Count Positive Cells and Calculate Viral
10. randed RNA adjuvant protects mice and ferrets against H5N1 avian influenza in oral delivery models Clin Vaccine Immunol 20 85 94 Xiong X et al 2012 The autophagy related gene 14 Atg14 is regulated by forkhead box O transcription factors and circadian rhythms and plays a critical role in hepatic autophagy and lipid metabolism J Biol Chem 287 39107 39114 Haidar M et al 2012 Integrin 281 mediates tyrosine phosphorylation of vascular endothelial cadherin induced by invasive breast cancer cells J Biol Chem 287 32981 32992 8 h CELL BIOLABS INC ae 10 11 12 13 14 15 16 17 18 19 Hisamitsu T et al 2012 Na H exchanger 1 directly binds to calcineurin A and activates downstream NFAT signaling leading to cardiomyocyte hypertrophy Mol Cell Biol 32 3265 3280 Lee S et al 2012 Adiponectin abates diabetes induced endothelial dysfunction by suppressing oxidative stress adhesion molecules and inflammation in type 2 diabetic mice Am J Heart Circ Physiol 303 H106 H115 Polling J et al 2011 Induction of smooth muscle cell migration during arteriogenesis is mediated by Rap 2 Arterioscler Thromb Vasc Biol 31 2297 2305 Triulzi C et al 2010 Antibody dependent natural killer cell mediated cytotoxicity engendered by a kinase inactive human HER2 adenovirus based vaccination mediates resistance to breast tumors Cancer Res 70 7431 7441 Peled M et
11. s for all materials containing BSL 2 organisms Preparation of Reagents The table below is suggested for tests in a 24 well plate Use twice the amount of reagents for samples in a 12 well plate e 1X Anti Hexon antibody solution Prepare a 1X anti hexon antibody solution by diluting the provided 1000X Anti Hexon antibody stock 1 1000 in 1 BSA PBS Store the diluted solution on ice e 1X Secondary antibody solution Prepare a 1X Secondary antibody solution by diluting the provided 1000X stock 1 1000 in 1 BSA PBS Store the diluted solution on ice e 1X DAB working solution Prior to use FRESHLY prepare a 1X DAB working solution First dilute the provided 10X Diluent to 1X with ddH O and add H5O to a final concentration of 0 01 Then dilute the 25X DAB stock to 1X with 1X Diluent H202 and use the 1X DAB working solution immediately Note When dilute 10X diluent use ddH O Heavy metals in impure H5O will cause DAB precipitation 20 tests Reagents 24 well plate 1000X Anti hexon Antibody 5 uL 1000X Secondary Antibody 5 uL 25X DAB 200 uL Final Volume Each Reagent 5mL Table 1 Preparation of antibody and DAB solutions CELL BIOLABS INC ae Preparation of Adenoviral Samples 1 Immediately before infection create a 10 fold serial dilution of viral sample from 10 to 107 First dilute original viral sample 1 100 For example adding 10 uL of viral sample to a sterile tube containing 990 uL o
12. urchaser 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 2013 2015 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing e CELL BIOLABS INC P A
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