BioPORTER Reagent
Contents
1. 4 Stability And Storage naserie satker Eiai EiS aea ENE SE TaN EAEN 4 Introd cti oN enstanen e e a E EE E EEA EEA ER ANR 5 Summary of the BioPORTER Protein Delivery Mechanism 6 METHODS AND PROCEDURES General Protocol Preparation of BioPORTER Protein complexes 7 Example Protocols Delivery of fluorescent antibody B galactosidase or dextran 9 Delivery of granzyme B and caspase 3 into Jurkat amp Ki Ras 267 B1 615 10 APPENDIX Protocol for Optimization 12 Quality Controleer e E 13 Troubleshooting Guide 4 44224414 84144 11 E R E 011 14 Quick reference Protocol for Experienced 15 Recordkeeping 1 2 42244124120441141188 8 O 16 BioPORTER QuikEase Genlantis a division of Gene Therapy Systems Inc Rev 03152006 3 Ph 888 428 0558 e www genlantis com OVERVIEW Kit Contents and Ordering Information The BioPORTER reagent is the latest innovation in macromolecule delivery2. Diagram Depicting Protein Delivery into Cells by BioPORTER Reagent Cell Membrane Lipid bilayer R R R Nucleus BioPORTER Protein Complexes BioPORTER QuikEase Genlantis a division of Gene Therapy Systems Inc Rev 03152006 6 Ph 888 428 0558 e www genlantis com NOTE METHODS AND PROCEDURES 1 General Protocol The conditions that follow are recommended only as starting guidelines For best performance of the BioPORTER reagent we recommend that you optimize component concentration cell number time of incubation and protein hydration buffers Because optimum conditions are cell type and assay dependent we are providing you with optimization guidelines in the Appendix section on page 12 Table 2 Suggested number of reactions for each QuikEase tube of BioPORTER Tissue Culture Dish Number of reactions tube 96 well 10 24 well 4 12 well 2 6 well 1 Each BioPORTER reagent QuikEase tube was designed for one single use per well in a 6 well plate If other tissue culture dishes are preferred prepare the BioPORTER protein complexes as we recommend for the 6 well plate format and divide the mixture among as many wells as needed For example 1 QuikEase tube can be used in 4 wells of a 24 well plate 1 1 Preparation of the BioPORTER Protein Complexes A 7 Dilute protein in buffer OC 2 Add protein solution to tube containing dry f
3. e Increase the BioPORTER reagent hydration volume e Lower the concentration of protein or biomolecule used Quality Control To assure the performance of each lot of the BioPORTER reagent we qualify each component using rigorous standards The following assays are conducted to qualify the function and activity of each kit component in living cells Kit Component Quality Control Standard BioPORTER 1 Efficient FITC antibody delivery in NIH 3T3 cells reagent 2 Efficient B galactosidase delivery in NIH 3T3 cells 3 Induction of apoptosis in Jurkat cells using granzyme B Delivery efficiency is assayed by monitoring the percentage of cells that become apoptotic by flow cytometry see example protocols 4 Testing for absence of bacterial and fungal contaminants FITC Antibody 1 Analysis by gel electrophoresis and measurement of fluorescence Positive Control 2 Testing for intracellular delivery by BioPORTER reagent in NIH 3T3 cells B galactosidase 1 Testing for intracellular delivery by BioPORTER reagent in NIH 3T3 cells Positive Control BioPORTER QuikEase Genlantis a division of Gene Therapy Systems Inc Rev 03 15200615 Ph 888 428 0558 www genlantis com Troubleshooting Guide Problem Possible Causes Recommended Solutions Low delivery Suboptimal protein peptide Titrate the concentration and the hydration volume of BioPORTER efficiency concentration Suboptimal hyd
4. protein Dish Medium Mix 96 well 1 2 x 10 50 50 24 well 0 5 1 x 10 125 ul 125 ul 12 well 1 2x 10 250 ul 250 ul 6 well 2 4 x 10 500 ul 500 BioPORTER QuikEase For adherent cells directly add the BioPORTER protein complexes resuspended in serum free medium onto the cells For suspension cells count the cells centrifuge them at 1200 rpm for 5 minutes then resuspend them in the appropriate volume of serum free medium see Table 5 above Adjust their concentration according to the size of your plate Pipette the BioPORTER protein mixtures into the tubes with cells and transfer all to wells dishes 1 1 6 Incubate for 3 4 hours at 37 C If longer incubation time is required add one volume of 20 serum containing medium directly to the well or dish It is not necessary to change the medium up to 24 hours after the initial serum free incubation Replace medium as required for longer incubations times The presence of serum in the first hours of incubation inhibits efficient delivery Make sure that the first 3 4 hours of incubation is done in serum free conditions followed by growth in serum containing medium 1 1 7 Proceed with your experiment for observation or detection assays Cells can be fixed or can be observed alive 1 1 8 Example protocols for the two positive controls included in the kit fluorescein labeled antibody and B galactosidase are provided below 2 Example Protocols 2 1 De
5. Fax 858 623 9494 or 858 558 3617 E mail Orders genlantis com Web Site http www genlantis com Stability and Storage The BioPORTER reagent QuikEase tubes are shipped frozen Upon receipt and for long term use store all QuikEase tubes or kits at 20 C The BioPORTER reagent is stable for at least 1 year at the recommended storage temperature BioPORTER QuikEase Genlantis a division of Gene Therapy Systems Inc Rev 03152006 Ph 888 428 0558 e www genlantis com Introduction Congratulations on your purchase of the BioPORTER reagent QuikEase kit The BioPORTER reagent is a new versatile and efficient reagent for intracellular delivery of bioactive molecules such as proteins peptides or antibodies into a broad range of cell types Although there are many transfection reagents available to introduce transcriptionally active DNA into viable cells approaches to deliver functional peptides and proteins into living cells are limited For this reason Gene Therapy Systems Inc has developed a different protein delivery approach using a unique lipid based carrier system The resulting BioPORTER is a novel reagent that contains a proprietary reactive lipid mixed with other components The BioPORTER reagent is easy to use and more economical than both microinjection and electroporation for delivering biologically active proteins into living cells The specific formulation of BioPORTER can deliver various molecules ove
6. the amount of protein per well can be divided by 2 4 and 10 for 12 24 and 96 well plates respectively IMPORTANT Our experimental results suggest that some highly positively charged molecules interact poorly with the BioPORTER reagent and are therefore not delivered into cells efficiently However this is not a general rule since granzyme B highly positively charged at neutral pH is delivered effectively 1 1 3 Use 40 ul of the diluted protein solution to hydrate one QuikEase tube containing the dried BioPORTER reagent Hydration volume can vary between 20 and 100 ul according to your desired protein concentration Pipette up and down 3 to 5 times Let stand at room temperature for 5 minutes then vortex gently and briefly 3 5 seconds at a low to medium speed 1 1 4 Bring the final volume of BioPORTER protein mixture to 0 5 ml with serum free medium 1 1 5 Aspirate the medium from the cells to be tested wash once with serum free medium optional and add the appropriate volume of serum free medium to the well see Table 5 Transfer the appropriate volume of the BioPORTER protein mix onto cells see Table 5 BioPORTER QuikEase Genlantis a division of Gene Therapy Systems Inc Rev 03152006 8 Ph 888 428 0558 e www genlantis com NOTE NOTE Table 5 Suggested Cell Numbers and BioPORTER protein Volumes Well Tissue Culture Number of Cells Volume of Serum free BioPORTER
7. and grow overnight For Jurkat cells see 2 2 5 below 2 2 2 Dilute caspase 3 to 330 660 and granzyme B to 15 45 ng ul in HBS buffer formulas are available in section 1 1 2 2 3 Hydrate one QuikEase tube with 40 ul of the diluted protein solution Pipette up and down 3 to 5 times Incubate at room temperature for 3 5 minutes then vortex briefly and gently at low to medium speed for a few seconds 2 2 4 Bring the final volume of the BioPORTER Protein mix to 0 5 ml with serum free medium BioPORTER QuikEase Genlantis a division of Gene Therapy Systems Inc Rev 0315200610 Ph 888 428 0558 e www genlantis com 2 2 5 2 2 6 2 2 7 2 2 8 For adherent cells like Ki Ras 267 81 aspirate the medium from cells wash once with serum free medium optional and add 125 ul of serum free medium to the well Transfer 125 ul of the BioPORTER protein mix directly onto the cells enough for 4 wells of a 24 well plate For suspension cells like Jurkat count and pellet the cells resuspend them in 125 ul of serum free medium at 8 x 10 cells ml Pipette 125 of the BioPORTER Protein mix into the 125 of cell suspension and then transfer the whole mixture to a 24 well plate Incubate cells in a 5 CO incubator at 37 C for 4 hours then add 1 ml of serum containing medium directly to the wells and incubate overnight Proceed with an apoptosis assay using any commercially available annexin V propidium
8. incubation time needs to be longer than 4 hours 10 After incubation wash cells and proceed with the appropriate assay Delivery of Apoptotic proteins granzyme B caspase 3 or caspase 8 1 2 Seed 0 5x10 adherent cells in 24 well plates and culture overnight For suspension cells see step 5 below Dilute caspase 3 at 330 0 1 units ul and granzyme B at 45 in HBS Use galactosidase as a negative control by diluting it to 0 1 ug ul in PBS Add 40 ul of the diluted protein solution to the BioPORTER dry film and mix by pipetting up and down 3 to 5 times Incubate at room temperature for 3 5 minutes Vortex BioPORTER protein complexes briefly then bring up final volume to 500 with serum free medium e For adherent cells bring the final volume to 500 ul with serum free medium Aspirate the medium from the cells to be tested add 125 of serum free medium to the cells and then transfer 125 of the BioPORTER protein mixture directly onto the cells enough for 4 wells e For suspension cells count and pellet the cells resuspend them in serum free medium at 8 x 10 cells ml Pipette 125 of the BioPORTER protein mixture to 125 ul of the cell suspension and then transfer it to a 24 well plate Incubate cells in a 5 CO incubator at 37 C for 4 hours then add 1 2ml of 10 serum containing medium directly to the well and incubate overnight The next day proceed with the apoptosis a
9. is effective in multiple cell types No cytotoxicity Fast transduction of macromolecules 3 to 4 hours post delivery Reagent that does not rely on covalent bonds Extended stability with long shelf life at 20 C Table 1 Cell Types Successfully Tested with BioPORTER HeLa S3 Jurkat HeLa BHK 21 293 Ki Ras 267 B1 HepG2 CHO K1 NIH 3T3 COS 7 P19 CV 1 B16 F0 K562 MDCK COS 1 BioPORTER QuikEase Genlantis a division of Gene Therapy Systems Inc Rev 03152006 5 Ph 888 428 0558 www genlantis com Summary of the BioPORTER Protein Delivery Mechanism The dried BioPORTER reagent is directly formulated with a solution of the protein or peptide to be delivered The BioPORTER reagent reacts quickly and interacts non covalently with the protein peptide or other molecules creating a protective vehicle for immediate delivery into cells The hydrated mixture is then added onto cells and the BioPORTER protein complexes attach to negatively charged cell surfaces The BioPORTER reagent capthen fuse directly with the plasma membrane and deliver the captured protein into cells coo QD in Figure 1 or the BioPORTER protein complexes are endocytosed and then fuse with endosomes releasing the BioPORTER captured proteins into the cytoplasm see in Figure 1 Delivery of molecules with the BioPORTER reagent is very easy and requires only 4 hours of incubation in serum free condition with the target cells Figure 1
10. technology It is a unique lipid based formulation that allows the delivery of proteins peptides or other bioactive molecules into a broad range of cell types The BioPORTER QuikEase kits contain 24 or 96 individual tubes of the BioPORTER reagent in a convenient ready to use single reaction format Each tube contains sufficient material of the lyophilized BioPORTER reagent to perform 1 reaction in a 6 well plate or 35mm dish or 4 reactions in a 24 well plate or 16mm wells The BioPORTER reagent is ready to use and only needs to be formulated with the protein solution or other molecule of interest to be delivered according to section 1 1 on page 7 Catalog Number Description Size or Aliquot Number of Tubes BP502424 24 BioPORTER QuikEase Reagent 1 reaction per tube lyophilized B galactosidase control protein 10 ug at 100 ug ml 1 FITC antibody control protein 10 ug at 100 ug ml fluorescein labeled goat IgG BP509696 96 BioPORTER QuikEase Reagent 1 reaction per tube lyophilized B galactosidase control protein 10 ug at 100 ug ml 1 FITC antibody control protein 10 ug at 100 ug ml fluorescein labeled goat IgG Use the contents of the table above to determine the appropriate catalog number for your needs You can order the products above by contacting us at Gene Therapy Systems Inc 10190 Telesis Court San Diego CA 92121 Phone 888 428 0558 U S Toll free or 858 457 1919
11. BioPORTER Reagent QuikEase Single Use Tubes Instruction Manual Catalog Numbers BP502424 BP509696 Z Genlantis Purchaser Notification Limited License The purchase price paid for the BioPORTER Protein Transfection Reagent by end users grants them a non transferable non exclusive license to use the reagent and its components as listed in the Contents section This reagent is intended for internal research only by the purchaser Such use is limited to the delivery or transfection of proteins into cells as described in the product manual Furthermore research only use means that this kit and all of its contents are excluded without limitation from resale repackaging or use for the making or selling of any commercial product or service without the written approval of Genlantis a division of Gene Therapy Systems Inc Genlantis Separate licenses are available from Genlantis for the express purpose of non research use or applications of the BioPORTER reagent To inquire about such licenses or to obtain permission to transfer or use the enclosed material contact the Director of Licensing at Genlantis There is a Patent Pending on the BioPORTER reagent Purchasers may terminate this License at any time by returning all BioPORTER reagent and or kit material and documentation to Genlantis or by destroying all BioPORTER reagent or kit components Purchasers are advised to contact Genlantis with the notification that
12. BioPORTER Protein Mix 1 N Dilute protein peptide or molecules of choice in HBS or PBS buffer Concentration depends the molecules used 50 250 ug ml is suggested Add 40 ul of the diluted protein solution directly to BioPORTER dry film and mix by pipetting Incubate at room temperature for 3 5 minutes Vortex BioPORTER protein mix briefly then add 0 5ml of serum free medium Transfer the appropriate volume of the mixture onto cells see Table 5 in section 1 1 5 Incubate for 4 hours Add serum containing medium if cells continue to incubate longer than 4 hours Example Protocols gt 7 9 gt Galactosidase or FITC Ab delivery in a 24 well plate 22 mm cover slips Seed 0 5 1x10 cells in 24 well plate or on cover slips and let grow overnight Dilute 4 8 ug of protein in 40 of HBS Ab or PBS B Galactosidase Hydrate BioPORTER dry film with 40 ul of the diluted protein solution and mix by pipetting up and down 3 to 5 times Incubate at room temperature for 5 minutes Vortex BioPORTER protein complex briefly then bring up final volume to 500 with serum free medium Blot dry coverslips and put in 35 mm dish or for 24 well plates aspirate old medium and add 125 of serum free medium to the cells Transfer 125 ul of the BioPORTER protein medium mixture to each well Incubate cells in a 5 CO incubator at 37 C for 4 hours Add serum containing medium if
13. a BioPORTER reagent is being returned in order to obtain a refund and or to expressly terminate a research only license granted through the purchase of the kit s This document covers in full the terms of the BioPORTER research only license and does not grant any other express or implied license The laws of the State of California shall govern the interpretation and enforcement of the terms of this License Product Use Limitations The BioPORTER reagent and all of its components are developed designed intended and sold for research use only They are not to be used for human diagnostic or included used in any drug intended for human use All care and attention should be exercised in the handling of the kit components by following appropriate research lab practices For more information or for any comments on the terms and conditions of this License please contact Director of Licensing Genlantis a division of Gene Therapy Systems Inc 10190 Telesis Court San Diego CA 92121 Phone 888 428 0558 U S Toll free or 858 457 1919 Fax 858 623 9494 or 858 558 3617 Email licensin enlantis com BioPORTER QuikEase Genlantis a division of Gene Therapy Systems Inc Rev 03152006 5 Ph 888 428 0558 www genlantis com TABLE OF CONTENTS Page OVERVIEW Purchaser Notification iszccssccciscevsccaseevsscadecoddcagacbccagnevccassevascadeseddagauebviatgatecsasooti cendoondasmaenacea geass 2 Kit Contents and Ordering Information
14. ilm of BioPORTER reagent 3 Incubate for 5 minutes at room temp and vortex 2 4 Add serum free medium 5 Add to cells and incubate BioPORTER QuikEase Genlantis a division of Gene Therapy Systems Inc Rev 03152006 7 Ph 888 428 0558 www genlantis com 1 1 1 Dilute proteins peptides or other molecules in one of the following buffers HBS 10 mM Hepes 150 mM NaCl pH 7 0 PBS 20 mM Na phosphate 150 mM NaCl pH 7 4 The final concentration of your molecules of interest will vary according to their intrinsic properties and the type of assay performed Further optimization guidelines are offered in the Appendix section on page 12 Table 3 below lists ranges of concentrations for various molecules that resulted in good delivery Table 3 Protein Concentration Ranges Used Successfully for Delivery with BioPORTER Antibody B gal or dextran sulfate 50 250 pg ml Caspase 3 0 05 0 3 units ul 165 to 1000 Granzyme B 7 5 to 60 1 1 2 The amount of protein or other molecules to be delivered will depend on the type of experiment cell type assay sensitivity plate size etc See Table 4 below for suggested amounts Table 4 Protein Amounts Used Successfully for Delivery with BioPORTER Culture Dish Ab or B gal ug Caspase3 ng Granzyme B ng 6 well 5 10 10 20 500 2000 If other tissue culture dishes are preferred
15. iodine labeling kit This assay can also be done at earlier time points Below is a brief protocol for a common apoptosis assay 1 Transfer medium and cells after very mild trypsinization for adherent cells to 13 x 75mm plastic tubes Wash wells with some serum containing medium pool them together and centrifuge at 1400 rpm for 5 minutes 2 Wash cells with 500 cold PBS without disturbing the pellet Centrifuge at 1000 rpm for 3 minutes Resuspend cells in 100 ul of cold annexin V binding buffer 4 Add annexin V FITC and propidium iodine PI to your samples and incubate at room temperature according to the annexin V PI labeling kit manufacturer protocol 5 Analyze your samples as soon as possible by flow cytometry or fluorescence microscopy BioPORTER QuikEase Genlantis a division of Gene Therapy Systems Inc Rev 03152006 11 Ph 888 428 0558 www genlantis com APPENDIX Protocol for Optimization It is highly recommended that you optimize your reaction conditions in order to get the best BioPORTER reagent performance Following are the many parameters that can be optimized Amount of protein peptide or other molecules to be delivered Hydration buffer containing the diluted protein solution Concentration of the protein solution during the preparation of the complexes Amount of BioPORTER reagent delivered to cells Hydration volume for BioPORTER reagent Cell types and cell culture dens
16. ity Time of incubation Many of these factors have been investigated by us during the development of the BioPORTER reagent We recommend that you optimize one parameter at a time using the suggested conditions in the Methods and Procedures section Start by using a fixed amount of BioPORTER reagent i e use one BioPORTER QuikEase tube per well 6 well plate or 1 4 of a QuikEase tube of BioPORTER per well 24 well plate Vary the amount of protein to be delivered Use a standard buffer like HBS or PBS for dilutions Depending on the sensitivity of the endpoint assay a greater amount of protein may be required If further optimization is required fix the concentration and amount of protein peptide to be delivered and vary the volume of BioPORTER protein mix transferred to cells see Table 6 below BioPORTER will interact with your molecules of interest via hydrophobic and electrostatic interactions and because each molecule will have different charge and hydrophobicity the amount of BioPORTER may need to be changed Although BioPORTER is not cytotoxic at the recommended concentrations it may show some signs of cytotoxicity at higher reagent to cells concentration ratios Table 6 BioPORTER protein Mix Volume Ranges Per Well Tissue Culture Plate Sizes BioPORTER protein Mix Volume Range ul 96 well 35 75 24 well 50 300 12 well 125 500 6 well 250 500 After identification of the correc
17. livery of Fluorescent antibody B galactosidase or dextran sulfate 2 1 1 Seed 2 4 x 10 cells well in a 6 well plate or 0 5 1 x 10 cells well in a 24 well plate or on cover slips and let grow overnight 2 1 2 Dilute 4 8 ug of FITC Ab dextran sulfate or B galactosidase in 40 ul of HBS or PBS For f galactosidase we recommend using PBS buffer formulas available in section 1 1 The FITC Ab and B galactosidase provided in the kit are ready to use without further dilution Just thaw and mix well the positive controls before use 2 1 3 Hydrate one QuikEase tube with 40 ul of the diluted protein solution Pipette up and down 3 to 5 times Incubate at room temperature for 3 5 minutes then vortex briefly and gently at low to medium speed for a few seconds 9 Genlantis a division of Gene Therapy Systems Inc Rev 03152006 Ph 888 428 0558 e www genlantis com 2 1 4 Bring the final volume of the BioPORTER protein mix up to 0 5 ml with serum free medium 2 1 5 Aspirate the medium from the cells wash once with serum free medium optional and add the appropriate volume of serum free medium to wells see Table 5 Transfer the appropriate volume of the BioPORTER protein mix onto the cells see Table 5 e For a 6 well plate directly transfer the total volume of BioPORTER protein complexes 0 5 ml to each well e For a 24 well plate transfer 125 ul of the mixture per well Consequently 4 wells can be assayed Similarl
18. ly prepared BioPORTER protein complexes should be freshly prepared If complexes have been prepared and stored for too long aggregation may occur High amount of protein used Too much protein or too high of a could cause aggregation Lower the concentration or the amount of protein to be delivered Cytotoxicity Excess BioPORTER used Decrease the amount of reagent used Molecules delivered are toxic Use the appropriate control reactions like cells alone BioPORTER alone control or safe protein alone and compare to when formulated with the BioPORTER reagent Check the purity of the molecule of interest to be delivered Unhealthy cells Check cells for contamination Thaw a new batch of cells Cells are too confluent or cell density too low Check the culture medium pH kind used last time changed etc Check materials used for proper function culture plates incubator temperatures etc For additional troubleshooting assistance please contact our Technical Support Department Toll free number 888 428 0558 extension 1 E mail techl genlantis com BioPORTER is a registered trademark of Gene Therapy Systems Inc BioPORTER QuikEase Genlantis a division of Gene Therapy Systems Inc Rev 03152006 14 Ph 888 428 0558 e www genlantis com Quick Reference Protocol for Experienced Users General Protocol Preparation of
19. r a broad range of cell types in serum free conditions Molecule delivery is fast and reaches optimum levels after 4 hours of incubation Various molecules such as fluorescent antibody high and low molecular weight dextran sulfate phycoerythrin BSA f galactosidase caspase 3 caspase 8 and granzyme B have been successfully delivered into the cytoplasm of a variety of different adherent and suspension cells with the BioPORTER reagent Furthermore apoptotic proteins like granzyme B caspase 3 or caspase 8 that were delivered into cells remained functional and drove cells into apoptosis With BioPORTER you can make your macromolecules directly available for a variety of studies like intercellular signaling cell cycle regulation control of apoptosis study of oncogenesis and transcription regulation to name a few The BioPORTER protein transfection reagent has been extensively tested to verified its effectiveness in delivering active molecules into a wide variety of cells Table 1 The new QuikEase kits that contain single use tubes of the BioPORTER reagent make it a more convenient tool for functional genomics or proteomics With the efficiency of the BioPORTER reagent and the convenience of QuikEase format you get the following benefits BioPORTER pre coated tubes for significantly reduced reagent preparation time Single reaction size for greater speed accuracy and format flexibility Streamlined protocol to minimize contamination Reagent
20. ration buffers Change the protein dilution buffer and or the pH to improve the delivery Insufficient mixing Allow the mixtures to form for at least 3 minutes Mix well by molecules to be delivered BioPORTER and protein pipetting up and down Do not vortex vigorously at this step Suboptimal amount of Vary the amount of reagent added onto cells as suggested in the BioPORTER used optimization protocol Molecules to be delivered are Highly positively charged molecules are difficult to deliver with highly charged BioPORTER Modify the hydration buffer or pH to change the charge of the molecules Unknown properties of the Mix a fluorescent molecule or directly label the protein of interest in order to monitor delivery Suboptimal cell density Use cells that are 50 60 cells confluent Wrong medium used Make sure to use serum free medium during the first hours of delivery Improper storage BioPORTER reagent is very stable but long exposure to elevated temperatures may cause degradation of the reagent Store BioPORTER at 20 C Suboptimal incubation time Incubate BioPORTER protein complexes with cells for at least 3 4 hours Type of cell line used is difficult to transduce Test BioPORTER with the positive controls in parallel with cell lines that were successfully used see Table 1 on page 5 for cell line suggestion Aggregation BioPORTER protein complexes not fresh
21. ssay BioPORTER QuikEase Genlantis a division of Gene Therapy Systems Inc Rev 03152006 gt Ph 888 428 0558 e www genlantis com Record Keeping Table The table below is provided for your convenience Photocopy and use it to keep track of all parameters that are necessary for efficient delivery of your biomolecules of interest using the BioPORTER reagent Protein Name Buffer Name Protein Amount Protein Concentration Hydration Volume Type of Cells Number of Cells Well BioPORTER protein Mix Volume Incubation Time Efficiency of delivery
22. t amount of BioPORTER and protein to be used you can then optimize the volume used to hydrate the BioPORTER dry film step 1 1 3 with the protein solution To test this parameter fix the protein amount and vary the hydration volume for BioPORTER from 20 to 100 ul For more flexibility you might want to consider using the standard one vial configuration of the BioPORTER reagent offered under catalog number BP502401 BioPORTER QuikEase Genlantis a division of Gene Therapy Systems Inc Rev 03152006 12 Ph 888 428 0558 e www genlantis com 5 Different protein dilution buffers like Tris HBS and PBS can be tested For some molecules we have found that the buffer composition may be critical For example B galactosidase delivery efficiency is very good with PBS but not with Tris for dextran sulfate HBS works best pH may also be critical for some molecules because of their different charge and hydrophobicity Varying the pH may help improve interaction with the BioPORTER reagent 6 At this point the cell number can also be optimized since delivery efficiency may be sensitive to the confluency of cells in culture 7 Depending on the type of functional assay performed shorter or longer incubation times may be necessary If aggregation of the BioPORTER protein complexes occurs during optimization seen as large glowing particles try one or more of the following recommendations e Briefly sonicate the BioPORTER protein mix
23. y 2 and 10 wells can be tested for 12 and 96 well plates respectively 2 1 6 Incubate cells in a 5 CO incubator at 37 C for 4 hours Add 1 volume of 20 serum containing medium directly to the well if incubation time needs to be longer than 4 hours 2 1 7 After incubation wash the cells twice with PBS and proceed to assay e Fluorescent microscopy after washing mount cells that are growing on cover slips directly onto a hanging drop slide with PBS Living cells are then directly observed under a microscope Alternatively cells can be fixed for observation e galactosidase assay X Gal staining for 6 well plates for all of our assays we have used the Genlantis X Gal staining Kit cat A10300K with the following brief protocol Aspirate medium 4 to 24 hours after B galactosidase delivery Wash cells twice with PBS 2ml Fix cells with the 1X fixing solution 1ml for 10 minutes at room temperature Prepare staining solution Remove fixing solution and gently wash cells 2 times with PBS 2ml Add staining solution 1ml and incubate 2 hours to overnight at 37 C Remove staining solution wash cells with PBS and examine under a light microscope Calculate percentage of stained cells if desired SUS 2 2 Delivery of Granzyme B and Caspase 3 Into Jurkat or Ki Ras 267 1 Cells 2 2 1 For adherent cells like Ki Ras 267 B1 prostate cancer seed 0 5 x 10 well 24 well plate
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