Adeno-X™ Rapid Titer User Manual
Contents
1. These photos were taken using a 5X objective Clontech Laboratories Inc www clontech com Protocol No PT3651 1 ATakara Bio Company Version No 081012 4 Adeno X Rapid Titer Kit User Manual ll List of Components Store Rat Anti Mouse Antibody HRP conjugate and Stable Peroxidase Buffer at 4 C DO NOT FREEZE Store Mouse Anti Hexon Antibody and 10X DAB Substrate at 20 C The following reagents are suitable for 60 titrations with 5 x 12 well plates or for 120 titrations with 5 x 24 well plates 60 ul Rat Anti Mouse Antibody HRP conjugate 30 ml 1X Stable Peroxidase Buffer 30 ul Mouse Anti Hexon Antibody 3 0 ml 10X DAB Substrate Ill Additional Materials Required The following materials are required but not supplied Phosphate buffered saline PBS pH 7 5 Phosphate buffered saline 1 bovine serum albumin PBS 1 BSA Dissolve 5 g Bovine serum albumin Sigma Fraction V Cat No A 3803 in 500 ml PBS above Store at 4 C 12 well culture plates BD Falcon Cat No 353043 Laminar flow hood BL2 Incubator humidified 5 CO2 Microscope with a 20X objective Hemacytometer Cell culture medium e g DMEM 10 fetal bovine serum antibiotics Methanol Optional BD Biocoat Collagen Type I 12 well plates BD Biosciences Discovery Labware Cat Nos 354500 amp 356500 Note Promotes stronger cell adhesion and helps prevent disruption of cell monolayer during rinses of the wells Op2. User Manual V Adeno X Rapid Titer Procedure continued Protocol 30 min hands on 3 hr overall KL Attention 10 B Protocol Fix Cells and Add Antibodies I Fix cells by VERY GENTLY adding 1 ml ice cold 100 methanol to each well Note Add methanol very gently Do this by adding the methanol to the wall of the well Do not dislodge the cell monolayer The mono layer can be easily dislodged until cells are fixed Using a Pipetman and not a 5 ml pipet can help This is particularly important for the 24 well plate Incubate the plate at 20 C for 10 min Aspirate methanol Gently rinse the wells three times with 1 ml PBS 1 BSA Dilute Mouse Anti Hexon Antibody 1 1 000 in PBS 1 BSA Aspirate final rinse from the wells Then add 0 5 ml of Anti Hexon Antibody dilution to each well Incubate 1 hr at 37 C on an orbital shaker orbital shaker optional Aspirate Mouse Anti Hexon Antibody Then gently rinse wells three times with 1 ml PBS 1 BSA Dilute Rat Anti Mouse Antibody HRP conjugate 1 500 in PBS 1 BSA Aspirate final rinse from the wells Then add 0 5 ml Rat Anti Mouse Antibody HRP conjugate dilution to each well Incubate 1 hr at 37 C on an orbital shaker orbital shaker optional Prior to removing the Rat Anti Mouse Antibody HRP conju gate prepare DAB working solution by diluting 10X DAB Sub strate 1 10 with 1X Stable Peroxidase
3. are comparable to values obtained with other infectious assay methods that normally take up to one week to perform The Adeno X Rapid Titer Kit takes advantage of the production of viral hexon protein in infected cells for the quantification of viral stocks Dilutions of the viral stock in question are used to infect HEK 293 cells Just 48 hours later these cells are fixed and stained with the antibody specific for the adenovirus hexon protein Signal is detected after a secondary antibody conjugated with horseradish peroxidase HRP amplifies the signal of the antihexon an tibody Figure 1 Subsequent exposure to metal enhanced DAB substrate turns only the infected cells dark brown Figure 2 Then the titer of the stock in question can be determined by counting the number of brown cells in a given area Each stained cell corresponds to a single infectious unit ifu This assay yields values that correlate well with plaque assay and gene transducing unit assay measurements as well as with OD measurements of total viral particles Bewig amp Schmidt 2000 For more information about different methods for adenoviral titration refer to ourAdeno X Expression System User Manuals which are available at www clontech com mamuals e Adeno X Expression System 1 User Manual PT3414 1 e Adeno X Adenoviral System 3 User Manual PT5177 1 Applications This kit has been developed for use with any adenoviral system provided that the hexon protein is bei
4. 1 Fix HEK 293 cells 2 Anti Hexon Antibody 3 HRP conjugated Antibody 4 Stain with DAB substrate Positive cells turn HEK 293 cell brown black Count positive cells amp calculate infectious units ifu 1hr 48 hr lt 3hr Figure 1 Adeno X Rapid Titer Method HEK 293 cells are infected with serial dilutions of adenovirus After the hexon proteins appear the cells are fixed and treated with a hexon protein specific antibody HRP conjugate antibody and developed with DAB Substrate During development the positive cells turn brown so that they can be easily counted under a 20X objective The ifu is calculated from the resulting average number of positive cells unit dilution Neg negative control 1The procedure to follow when using a 24 well plate is similar except for quantities please see Section V Adeno X Rapid Titer Procedure Protocol No PT3651 1 www clontech com Version No 081012 3 Clontech Laboratories Inc ATakara Bio Company Adeno X Rapid Titer Kit User Manual l Introduction amp Protocol Overview continued A Dilution of Adenoviral Stock Soln 10 33 B 10 4 C 1075 Figure 2 Fields of positive cells Different dilutions of adenovirus were used to infect HEK 293 cells and de veloped by the Adeno X RapidTiter method A cytopathic effect is evident in Panel A 10 In this example the 10 dilution Panel C would provide the field of cells most ideal for counting
5. Buffer you will need 500 ul DAB working solution per assay well Allow the 1X DAB working solution to come to room temperature Note Do not allow 10X DAB Substrate to come to room temperature Aspirate Rat Anti Mouse Antibody HRP conjugate dilution Gently rinse each well three times with 1 ml PBS 1 BSA 1 ml 0 5 ml 1 0 ml 0 5 ml 500 ul 1 ml 24 well 0 5 ml 0 5 ml 0 25 ml 0 5 ml 0 25 ml 250 ul 0 5 ml Protocol No PT3651 1 Version No 7 081012 www clontech com Clontech Laboratories Inc ATakara Bio Company Adeno X Rapid Titer Kit User Manual V Adeno X Rapid Titer Procedure continued C Protocol Develop Color and Quantitate 12 well 24 well 1 After removing the final PBS 1 BSA rinse add 500 pl DAB 500 ul 250 ul working solution to each well Incubate at room temperature for Protocol l min 30 min hands 2 Aspi dd 1 ml h well l l on Aspirate DAB and add 1 ml PBS to each well lm 0 5 m SG 3 Count a minimum of three fields of brown black positive cells using a microscope with a 20X objective and calculate the mean number of positive cells in each well Note Count dilutions with 10 or fewer positive cells An ideal field should contain 5 to 50 positive black brown cells Adjust your objective as needed See Figure 2 4 Calculate infectious units ifu ml for each well as follows infected cells field x fields well volume vi
6. E e thes o o Ss Clontech United States Canada 800 662 2566 Asia Pacific 1 650 919 7300 Europe 33 0 1 3904 6880 Japan 81 0 77 543 6116 Clontech Laboratories Inc ATakara Bio Company 1290 Terra Bella Ave Mountain View CA 94043 Technical Support US E mail tech clontech com www clontech com Adeno X Rapid Titer Kit User Manual Cat No 632250 PT3651 1 081012 Published 10 August 2012 Adeno X Rapid Titer Kit User Manual Table of Contents l Introduction amp Protocol Overview cccccccsssssssssecseceeeeseseeessseeaseeseeeeeeeeeeseesssensaneeseeseeneneess 2 IL List Of COMpPONENMHS cccceccesssseseeeceeceeeeeeseeeessensueeeseeseeeeeesseeeeaneuesesseseseesecseesesensaesesseseeseneess 5 Ill Additional Materials Reouired kee NENNEN ENEE 5 IV General Consideration i iic ccisicsccesccecieleciessssisvesuecoescetwcescesandevessantceteosccuadesesansevnseade lapeetesaseaatees 6 V Adeno X Rapid Titer Procedure cccccccccccccecscecsseessssssesssssseeeseeececeeeeeeeeseeseseeseseaasanaaaaaaaaesees 7 As Protocols Infect Cell ss eise ioc 2etesevisczaatsev aca a E xb bee s ege tee ee eege 7 B Protocol Fix Cells and Add Antbodies 8 C Protocol Develop Color and TE 9 Dx Protocol Example Calculation eege ee ea EEEE E A EG 9 Vi F u bleshooting Guide sessies raana inan EEEE AEE EEEE E EAAS 10 VIR TEE 10 Ais ING positives EE 10 B All cells are positive B
7. n question in diluent PBS or sterile medium For example Note Change filter tips at each serial dilution e 10 pl Adenoviral Stock diluted in 990 pl diluent 107 ml Add 100 pl to the 1 ml of cells e 100 pl of 10 dilution in to 900 pl of diluent 10 ml Add 100 pl to the 1 ml of cells 100 pl of 10 dilution in to 900 pl of diluent 10 ml Add 100 pl to the 1 ml of cells e 100 pl of 10 dilution in to 900 ul of diluent 10 ml Add 100 pl to the 1 ml of cells e 100 pl of 10 gt dilution in to 900 ul of diluent 10 ml Add 100 pl to the 1 ml of cells If you need to adjust the dilutions to 1 2 log increments it can be done as follows e 500 pl of 107 dilution in to 500 pl of diluent 5 x 10 ml Add 100 ul to the 1 ml of cells e 500 pl of 10 dilution in to 500 pl of diluent 5 x 10 ml Add 100 ul to the 1 ml of cells e 500 pl of 10 dilution in to 500 ul of diluent 5 x 10 ml Add 100 ul to the 1 ml of cells e 500 pl of 10 dilution in to 500 pl of diluent 5 x 10 ml Add 100 ul to the 1 ml of cells Notice to Purchaser Clontech products are to be used for research purposes only They may not be used for any other purpose including but not limited to use in drugs in vitro diagnostic purposes therapeutics or in humans Clontech products may not be transferred to third parties resold modified for resale or used to manufacture commercial products or to provide a service to third parties without written a
8. ng expressed The kit can be used to determine titers of the recombinant adenovirus created with our Adeno X Expression System 1 Cat No 631513 orone of our Adeno X Adenoviral Systems 3 Cat Nos 632264 632265 632266 632267 632268 632269 and 631180 Adeno X Expression Vectors carry a deletion in the El region of the adenovirus genome A cell line complementing missing E1 elements is required for amplification and titration of the El deleted adenoviruses One of the most commonly used El region complementing cell lines is human embryonic kidney 293 HEK 293 cell line which carries integrated in its genome constitutively expressed human adenovirus type 5 E1 sequences Graham et al 1977 Aiello et al 1979 For more information on expression cassettes and the Adeno X Viral genome see our Adeno X Expression System and System 3 User Manuals which are available at www clontech com mamuals We recommend the use of our Adeno X 293 Cell Line Cat No 632271 but this kit can be used with any cell line that complements the E1 elements missing from our Adeno X Expression Vectors e g HEK 293 cells Clontech Laboratories Inc ATakara Bio Company www clontech com Protocol No PT3651 1 Version No 081012 2 Adeno X Rapid Titer Kit User Manual l Introduction amp Protocol Overview continued Seed 12 well plate with HEK 293 cells 5 X 10 ml 3 amp Infect cells LY SS Hexon proteins expressed HEK 293 cell
9. orrect preparation of DAB working solution e Did not use PBS 1 BSA for washing steps C Cell monolayer is disrupted or comes off during fixing step e Use collagen coated plates for growing cells See Additional Materials Required Section e Add methanol very gently to the well e Rinses not gentle enough Vil References Aiello L Guilfoyle R Huebner K amp Weinmann R 1979 Adenovirus 5 DNA sequences transcribed in transformed hu man embryo kidney cells HEK Ad5 or 293 Virology 94 460 469 Bowie B amp Schmidt W E 2000 Accelerated Titering of Adenoviruses Bio Techniques 28 87 1 873 Graham F L Smiley J Russel W C amp Nairn R 1977 Characterization of a human cell line transformed by DNA from human adenovirus type 5 J Gen Virol 56 59 72 Price J Turner D Cepko C 1987 Lineage analysis in the vertebrate nervous system by retrovirus mediated gene transfer Proc Natl Acad Sci USA 84 1 156 160 Protocol No PT3651 1 www clontech com Clontech Laboratories Inc Version No 081012 ATakara Bio Company 9 Appendix A Adeno X Rapid Titer Kit User Manual Diluting Adenoviral Stock Solutions There are many ways to make dilutions of adenoviral stocks One important factor in making the dilutions and infect ing the cells is to be consistent in the amount of volume added to the well 0 1 ml in our procedure Using filter tips make serial dilutions of the adenoviral stock i
10. pproval of Clontech Laboratories Inc Your use of these products and technologies is subject to compliance with any applicable licensing requirements described on the product s web page at http www clontech com It is your responsibility to review understand and adhere to any restrictions imposed by such statements Clontech the Clontech logo and Adeno X are trademarks of Clontech Laboratories Inc All other marks are the property of their respective owners Certain trademarks may not be registered in all jurisdictions Clontech is a Takara Bio Company 2012 Clontech Laboratories Inc This document has been reviewed and approved by the Clontech Quality Assurance Department Clontech Laboratories Inc ATakara Bio Company www clontech com Protocol No PT3651 1 Version No 081012 10
11. re accuracy Protocol 30 min A Protocol Infect Cells ponis 1 Seed 1 ml of healthy log phase HEK 293 cells 5 x 10 cells ml 5 x 10 cells 2 5 x 10 cells 48 hr in each well of a 12 well plate Use standard growth medium overall e g DMEM 10 FBS antibiotics 12 well 24 well Note Cells will not completely adhere to the plate during infection 2 Using PBS or medium as diluent prepare 10 fold serial dilu tions of your viral sample from 10 to 10 ml For example 10 10 10 10 10 107 See Figure 1 see Appendix A for suggestions on how to dilute adenoviral stock solutions Use a fresh pipet tip for each step in the dilutions Note To improve accuracy you may need to adjust dilutions to 5 x 10 5 x 10 etc depending on the expected viral titer see Appendix A 3 Add 100 pl of viral dilution dropwise to each well 100 pl 50 pl ZK Note Perform duplicate infections to ensure accurate assay results 4 Incubate cells at 37 C in 5 CO for 48 hr 5 Aspirate medium Allow cells to dry in hood Do not overdry Note While it is also possible to use the 96 well format this is not recommended because the geometry of the well together with infection kinetics and staining procedures can combine to cause issues with the consistency of the assay Clontech Laboratories Inc www clontech com Protocol No PT3651 1 ATakara Bio Company Version No 081012 6 Adeno X Rapid Titer Kit
12. rowns Black ccs scsi theen cries teins onnee eassa EAER EEE EEN 10 C Cell monolayer is disrupted or comes off during fixing step cecsccessseeseseeetseseseseeeeseecaeeeeesecateeeens 10 Appendix A Diluting Adenoviral Stock Solutions REENEN 11 List of Figures Figure L Adeno X Rapid Titer Method gute ue EES dE ENEE eae E 3 Figure 2 Fields of positive Cells sasirnane i EENEG dE E 4 List of Tables Table I Derivation of Area Counted in PieldelWicll 10 Contact Us For Assistance Customer Service Ordering Technical Support Telephone 800 662 2566 toll free Telephone 800 662 2566 toll free Fax 800 424 1350 toll free Fax 800 424 1350 toll free Web www clontech com Web www clontech com E mail orders clontech com E mail tech clontech com Protocol No PT3651 1 www clontech com Clontech Laboratories Inc Version No 081012 ATakara Bio Company 1 Adeno X Rapid Titer Kit User Manual l Introduction amp Protocol Overview The Adeno X Rapid Titer Kit provides a complete set of materials for the quantification of adenoviral stocks Titra tion of adenoviral stocks is important for maintaining consistency between experimental samples and achieving the correct level of expression Also when producing viral stocks it is important to know the titer of infectious particles for successful virus production Results are obtained much more quickly with this kit within 48 hr than with standard assays The values obtained
13. rus ml x dilution factor D Protocol Example Calculation e Mean positive cells field 10 at 10 gt dilution e Fields well 20X objective 594 fields Protocol e Amount dilution added 0 1 ml Therefore e ifu ml 10 cells field x 594 fields well 0 1 ml x 107 ml 5 94 X 10 ifu ml Note This example calculation is for a 12 well plate See Table below Table I Derivation of Area Counted in Fields Well Objective Lenses Eyepiece Lenses 10X Fields Well Total Magni Field Field Area 12 Well Plate 24 Well Plate 96 Well Plate fication Diameter mm area 3 8 cm area 2 0 cm area 0 32 cm Clontech Laboratories Inc www clontech com Protocol No PT3651 1 ATakara Bio Company Version No 081012 8 Adeno X Rapid Titer Kit User Manual VI Troubleshooting Guide A No positive cells After completing the protocol no brown or black cells can be observed in any wells at any dilution e Anti Hexon or Rat Anti Mouse Antibody HRP conjugate was inadvertently omitted G e Rat Anti Mouse Antibody HRP conjugate was inadvertently frozen Note The HRP enzyme is sensitive to freeze thaw cycles e Did not infect for a full 48 hr before fixing cells As a result beson expression did not reach detection threshold B All cells are positive Brown Black e Inadequate rinsing steps e Incorrect or insufficient dilution of adenovirus stock e Incorrect dilution of Rat Anti Mouse Antibody HRP conjugate or inc
14. tional Orbital Shaker We recommend the use of our Adeno X 293 Cell Line Cat No 632271 but this kit can be used with any cell line that complements the E1 elements missing from our Adeno X Expression Vectors e g HEK 293 cells Protocol No PT3651 1 Version No 5 081012 www clontech com Clontech Laboratories Inc ATakara Bio Company Adeno X Rapid Titer Kit User Manual IV General Considerations When gathering data for the Adeno X Rapid Titer Kit it is important that the counted fields be selected in an unbiased manner Therefore we recommend that you randomly select a minimum of three fields to count and that the counted fields contain 10 50 positive cells assuming that the distribution of infected cells is random over the entire well Fields with fewer positives 5 10 cells can be counted if you do so we suggest that you count more fields 6 10 to achieve the same degree of accuracy In addition the degree of error introduced in each serial dilution may affect the result Therefore in order to maximize the accuracy measure samples in duplicate Two important factors in making the dilutions and infecting the cells are to be consistent in the amount of viral dilution added to the well 0 1 ml in our procedure and to be sure to use a new pipet tip for each dilution V Adeno X Rapid Titer Procedure PLEASE READ ENTIRE PROTOCOL BEFORE STARTING Each dilution of virus should be assayed in duplicate to ensu
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