BioPORTER Reagent
Contents
1. 5 x 10 cells ml Pipette 200 ul of cell suspension into BioPORTER protein mixture then transfer to a 24 well plate 8 Incubate cells in a 5 CO incubator at 37 C for 4 hours then add 1 2 ml of 10 serum containing medium directly to the well and incubate overnight 9 The next day proceed with the apoptosis assay BioPORTER Reagent Genlantis a division of Gene Therapy Systems Inc Rev 03152006 16 Ph 888 428 0558 e www genlantis com2. optimize component concentration cell number time of incubation and protein hydration buffers Because optimum conditions are cell type and assay dependent we provide you with optimization guidelines in the Appendix section on page 13 1 1 Preparation of the BioPORTER Reagent 1 1 2 Dissolve each tube Summary diagram of BioPORTER Preparation Process containing the dry film of 7 I Add Chloroform to FOET tds SISPORTERS i 50 ul of methanol or Reagent and 2 Transfer chloroform Vortex for 10 SS Reiley amount to tube 20 seconds at top speed before each use a i wv CAUTION Handle chloroform with caution by wearing the appropriate protective clothing and using a well ventilated space or hood To avoid excessive evaporation of chloroform keep containers closed as much as possible 3 Air dry tube contents 1 1 3 Pipette the desired amount of BioPORTER reagent into an eppendorf tube see table below for suggested amount Be sure to dispense the BioPORTER solution to the bottom of the tube The amount of BioPORTER reagent needed varies depending on the type of experiment you are conducting cell type assay sensitivity plate size etc We recommend that you start by using 2 5 ul of BioPORTER reagent per reaction in a 24 well plate or 10 ul for a 6 well plate and vary the amount of protein to be delivered We highly recommended that you optimize the delivery conditions by varying the amount of protein pe
3. reagent tubes at 20 C BioPORTER reagent is stable at least for 1 year at the recommended storage temperature BioPORTER Reagent Genlantis a division of Gene Therapy Systems Inc Rev 03152006 4 Ph 888 428 0558 e www genlantis com Introduction We congratulate you on your purchase of the BioPORTER reagent 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 effective 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 investigated a unique protein delivery approach using a lipid based carrier system The resulting BioPORTER reagent is a novel composition that contains a proprietary reactive lipid mixed with other components This new approach is easy to use and more economical than both microinjection and electroporation for delivering biologically active proteins into living cells The specific formulation of the BioPORTER reagent can deliver various molecules over a broad range of cell types in serum free conditions The delivery is fast and the optimum delivery is usually reached after 3 to 4 hours of incubation Various molecules such as fluorescent antibody high and low molecular weight dextran sulfate phycoerythrin BSA B galacto
4. reagent immediately after dissolving in 250 ul of chloroform This will help prevent extensive evaporation of the solvent and causing variations in reagent concentration Dried BioPORTER reagent can be safely stored at 20 C for at least one year without any substantial loss of activity 1 1 6 At this stage you can proceed to the next step preparation of the BioPORTER protein complexes or store the tubes at 20 C until use 1 2 Preparation of the BioPORTER Protein Complexes 1 2 1 Dilute the protein peptide or other molecules Summary Diagram of the BioPORTER protein in one of the following buffers Preparation Process f HBS 10 mM HEPES 150 mM NaCl pH 7 0 H PBS 20 mM Na phosphate 150 mM NaCl pH 7 4 Tris Buffer 10 mM Tris 150 mM NaCl pH 7 0 I Dilute protein in buffer z 1 2 2 The final concentration of your proteins 2 Add pr rein doliis to a E H peptides or molecules of interest will vary tube containing dry film of y according to their intrinsic properties and the BIePORTER reagent F type of assay performed Further optimization guidelines are offered in the ONCE H Appendix section on page 13 room semp and vortex ev H For the following molecules listed in Table 3 27 below we have found that the following E concentration ranges yielded good delivery 4 Add serum free medium results l ew 5 Add to cells and incubate a SSS BioPORTER Reagent Genlantis a division of Gene Therapy Systems Inc Rev
5. under a Aspirate medium 4 to 24 hrs after B gal delivery f Wash cells twice with PBS 500 ul microscope Alternatively cells can Fix cells with the 1x fixing solution 250 ul for 10 be fixed for observation min at room temperature p galactosidase assay X Gal Prepare the staining solution staining for all of our assays we Remove the fixing solution and gently wash cells 2 have used the Gene Therapy Systems X Gal staining Kit cat x with PBS 500 pl Add the staining solution 250 ul and incubate 2 hrs to overnight at 37 C A10300K with the brief protocol Remove the staining solution Wash cells with on the left PBS and examine under a light microscope Calculate percentage of stained cells if desired 2 2 Delivery of a Fluorescent Antibody p galactosidase or dextran sulfate High and low Molecular Weights for a 6 well plate or 35 mm dish 2 21 Seed2 x 10 cells in 6 well plate and let grow overnight 2 22 Pipette 10 ul of BioPORTER reagent dissolved in chloroform or methanol into the bottom of each eppendorf tube Evaporate the solvent as described in section 2 1 2 above 2 2 3 Dilute 5 10 ug of protein in 50 100 ul of appropriate buffer as in section 2 1 3 above 2 2 4 Hydrate the BioPORTER reagent dry film with 50 100 ul of the diluted protein solution Pipette up and down 3 to 5 times Incubate at room temperature for 5 minutes then vortex briefly and gently at low to
6. your samples as soon as possible by flow sample protocol on the left cytometry or fluorescence microscopy BioPORTER Reagent Genlantis a division of Gene Therapy Systems Inc Rev 03152006 12 Ph 888 428 0558 e www genlantis com APPENDIX Protocol for Optimization It is highly recommended to optimize your conditions in order to get the best performance for the BioPORTER reagent Several parameters can be optimized as follows Amount of protein peptide or other molecules to be delivered Hydration buffer containing the diluted protein solution Amount of BioPORTER reagent Concentration of the protein solution during the preparation of the complexes Hydration volume for BioPORTER reagent Cell types and cell culture density Time of incubation Many of these factors have been investigated at Gene Therapy Systems during the research and 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 l 2 Start by using a fixed amount of the BioPORTER reagent for example use 2 5 ul of BioPORTER reagent per reaction in a 24 well plate Vary the amount of protein to be delivered Use a standard buffer to do so for example HBS or PBS Depending on the sensitivity of the endpoint assay a greater amount of protein and BioPORTER reagent may be required If further optimization is required fix the concentration and amount
7. 03152006 8 Ph 888 428 0558 e www genlantis com Table 3 Protein concentration ranges used successfully for delivery Antibody B galactosidase or dextran sulfate 50 250 ug ml Caspase3 0 05 to 0 3 units ul 165 to 1000 pg ul Granzyme B 7 5 to 60 ng ul IMPORTANT 1 2 3 1 2 4 1 2 5 1 2 6 Our experimental results suggest that some though not all highly positively charged molecules interact poorly with the BioPORTER reagent and are therefore not delivered into cells efficiently Use the diluted protein solution to hydrate the dried BioPORTER reagent The amount of protein peptide antibody 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 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 low to medium speed Table 4 Suggested Quantity of Proteins and Hydration Volumes Tissue Protein Protein Protein Hydration Culture Ab B gal Caspase3 GranzymeB Volume uJ for Dish ug ng ug BioPORTER 96 well 0 1 0 25 0 25 0 5 0 01 0 05 10 24 well 1 2 2 4 0 075 0 5 10 25 12 well 2 4 4 8 0 15 1 25 50 6 well 5 10 10 20 0 3 2 50 100 60mm 10 20 20 40 0 5 3 100 400 100mm 25 50 50 100 0 75 4 250 500 Add serum free medium to the BioPORTER protein complex to bring the final delivery volume up to the
8. 67 pl bring the final volume of the BioPORTER protein mixture to 200 ul with serum free medium Aspirate the medium from the cells to be tested wash once with serum free medium optional and then transfer the BioPORTER protein mixture directly onto the cells 2 3 6 For suspension cells such as Jurkat count and pellet the cells resuspend them in serum free medium to 0 5 x 10 cells ml Add 200 ul of the cell suspension to the BioPORTER Protein mixture and then transfer the mix to a 24 well plate Apoptosis Assay Sample Protocol Transfer medium and cells after mild trypsinization 2 3 7 Incubate cells in a 5 CO incubator at 37 C for 4 hours Add 1ml of serum containing medium directly to the well and incubate overnight 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 Wash cells with 500 ul of cold PBS without disturbing the pellet centrifuge at 1000 rpm for 3 minutes 2 3 8 The next day proceed with the Resuspend cells in 100 ul of cold annexin V binding apoptosis assay using any buffer commercially available annexin V Add annexin V FITC and propidium iodine PI to propidium iodine labeling kit This your samples and incubate at room temperature assay can also be at time points according to the instructions of the annexin V PI ii than 4 hours see brief labeling kit manufacturer SIEHT a Analyze
9. BioPORTER Reagent Instruction Manual Catalog Numbers BP502401 BP509604 3 Genlantis Genlantis A Division of Gene Therapy Systems Inc 10190 Telesis Court San Diego CA 92121 Phone 888 428 0558 US Toll Free e 858 457 1919 Fax 858 623 9494 e 858 558 3617 E mail Orders genlantis com Web Site http www genlantis com 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
10. ER reagent BioPORTER Reagent 13 Genlantis a division of Gene Therapy Systems Inc Rev 03152006 Ph 888 428 0558 e www genlantis com 6 Atthis point the cell number can also be optimized since the delivery efficiency may be sensitive to the confluency of the cells in culture 7 Depending on the type of functional assay performed shorter or longer incubation time may be necessary If aggregation of the BioPORTER protein complexes occurs during optimization seen as large glowing particles under the microscope try one or any combination of the following recommendations Quality Control Briefly sonicate the BioPORTER protein mixture Increase the BioPORTER hydration volume Lower the concentration of protein or molecule Lower the amount of BioPORTER reagent 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 efficiency in NIH 3T3 cells 3 Induction of apoptosis in Jurkat cells using granzyme B and caspase3 Delivery efficiency is assayed by monitoring the percentage of cells that become apoptotic through flow cytometry 4 Testing for absence of bacterial and fungal contami
11. The dried BIoPORTER reagent formulation is first dissolved in a solvent and aliquoted into small eppendorf tubes according to the type of assays conducted see Methods and Procedures After complete drying BioPORTER is 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 can thepfuse directly with the plasma membrane and deliver the captured protein into the cells see D in Figure 1 or the BIoPORTER protein complexes are endocytosed by the cells and then with the endosome releasing the BioPORTER captured protein into the cytoplasm see in Figure 1 Delivery of molecules with the BioPORTER reagent is very easy and requires only 4 hours of incubation with the target cells Figure 1 Diagram depicting protein delivery into cells b BioPORTER reagent EP Cell Membrane Lipid bilayer BioPORTER Reagent Genlantis a division of Gene Therapy Systems Inc Rev 03152006 6 Ph 888 428 0558 e www genlantis com METHODS AND PROCEDURES 1 General Protocol NOTE The conditions that follow are recommended as good starting guidelines only For best performance of the BioPORTER reagent we recommend that you
12. amounts recommended in Table 5 below Table 5 Suggested Cell Number and Transfection Volume Tissue Culture Number of cells Total Delivery Mix Dish Volume 96 well 1 2 x 10 100 pl 24 well 0 5 1 x 10 250 ul 12 well 1 2 x 10 500 pl 6 well 2 4 x 10 1 ml 60mm dish 5 10 x 10 2 5 ml 100mm dish 10 20 x 10 5 ml Aspirate medium from the cells to be tested wash once with serum free medium optional and then transfer the final delivery mix onto cells For adherent cells directly add the BioPORTER protein complexes resuspended in serum free medium onto the washed cells For suspension cells first count the cells centrifuge them at 1200 rpm for 5 minutes and then resuspend them in serum free medium Adjust their concentration according to the size of your plate or dish and your transfection volume Pipette the cell suspension into the tube of BioPORTER protein mixtures and then transfer it to your well or dish BioPORTER Reagent Genlantis a division of Gene Therapy Systems Inc Rev 03152006 9 Ph 888 428 0558 e www genlantis com NOTE 1 2 7 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 after the initial serum free incubation However if incubation times that are longer than 24 hours are necessary medium can be replaced as needed The presence of serum in the fi
13. 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 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 reagent 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 Reagent Genlantis a division of Gene Therapy Systems Inc Rev 03152006 2 Ph 888 428 0558 e www genlantis co
14. d down do not vortex vigorously at this step Charge of Molecules to be Highly positively charged molecules are difficult to deliver with BioPORTER delivered reagent Modify the hydration buffer or pH to change the charge of the molecules Unknown properties of Mix a fluorescent molecule or directly label the protein of interest in order to molecules to be delivered monitor delivery Cell Density Use cells that are 50 60 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 Store BioPORTER reagent at 20 C Time of incubation Incubate BioPORTER protein complexes with cells for at least 3 4 hours Type of cell line used Test BioPORTER reagent with the positive controls in parallel with cell lines that were successfully used see Table on page 5 for cell line suggestions Aggregation Amount of BioPORTER reagent Too much reagent could cause aggregation lower the amount of used BioPORTER reagent if you think you used an excess amount Evaporation of the Excessive evaporation of the dissolved BioPORTER reagent will change its BioPORTER stock solution concentration Titrate down or use lower amounts of reagent Storage of the BioPORTER BioPORTER protein complexes should be freshly prepared If complexes protein complexes have been prepared and stored for too long aggregat
15. d for at least 2 hours Optionally vacuum the BioPORTER reagent film for 2 additional hours Preparation of BioPORTER Protein Mix 1 Dilute protein peptide or molecules of choice in HBS or PBS buffer Concentration depends on the molecules used 50 250 ug ml is suggested 2 Add diluted protein solution directly to BioPORTER dry film and mix by pipetting Hydration volume depends on the plate or dish size see Table 4 in 1 2 3 3 Incubate at room temperature for 3 5 minutes 4 Vortex BioPORTER protein mix briefly then bring volume up with serum free medium to an appropriate level see Table 5 in section 1 2 4 5 Transfer the mixture onto cells 6 Incubate for 4 hours 7 Add serum containing medium if cells continue to incubate longer than 4 hours Example p Galactosidase or FITC Ab delivery in a 24 well plate 22 mm cover slips Protocols 1 Seed 0 5 1 x 10 cells per well in 24 well plate or on cover slips and let grow overnight 2 Aliquot 2 5 ul of BioPORTER dissolved in 250 ul of solvent into an eppendorf tube 3 Evaporate the solvent in a cell culture hood for at least 2 hours Optionally vacuum the BioPORTER reagent film for 2 additional hours 4 Dilute 1 ug of protein in 10 ul of HBS Ab or PBS f Galactosidase 5 Hydrate BioPORTER reagent dry film with 10 ul of the diluted protein solution and mix by pipetting up and down 3 to 5 times 6 Incubate at room temperature for 5 minutes 7 Vortex BIoPORTER protein complex briefly bring
16. ion may occur Cytotoxicity Excess BioPORTER reagent Decrease the amount of reagent used Molecules delivered are toxic Use the appropriate control reactions like cells alone BioPORTER reagent alone control or safe protein alone and your molecule s of interest alone 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 is 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 888 428 0558 extension 1 Fax 858 623 9494 E mail tech genlantis com BioPORTER is a registered trademark of Gene Therapy Systems Inc BioPORTER Reagent Genlantis a division of Gene Therapy Systems Inc Rev 03152006 15 Ph 888 428 0558 e www genlantis com Quick Reference Protocol for Experienced Users General Preparation of BioPORTER Reagent Protocol 1 Dissolve each BioPORTER reagent tube in 250 ul chloroform or methanol 2 Vortex for 10 seconds at high speed 3 Aliquot the appropriate volume of BioPORTER reagent to the bottom of eppendorf tubes 4 Evaporate the solvent in a cell culture hoo
17. m TABLE OF CONTENTS Page OVERVIEW Purchaser NISL 2 Kit Contents and Ordering Information esses enne 4 Stability And Storage rette aor ttd esee i i rte cantante Ua e aea ENE A HERR e EP Giese 4 Inionotilvim e M 5 Summary of the BioPORTER Protein Delivery Mechanism sees 6 METHODS AND PROCEDURES General Protocol Preparation of BioPORTER Reagent tentes 7 Preparation of BioPORTER Protein Complexes seeeeteeeees 8 Example Protocols Delivery of a Fluorescent Antibody B galactosidase or Dextran Sulfate for 24 well Plates sse eren nnns 10 Delivery of a Fluorescent Antibody B galactosidase or Dextran Sulfate for 6 well Plates sess eene enne 10 Delivery of Granzyme B and Caspase 3 Into Jurkat amp Ki Ras 267 B1 Cells sess enne ennenenn 11 APPENDIX Protocol for Optimization esssssssssesseseeee eene nennen enne nennen tenente nenne nnn nnns 13 Quality Control M 14 Troubleshootitig Guide 5 2 tr ree rd Re RE REA ER TWIN EA are EORR eR NR dU PEERS 15 Quick reference Protocol for Experienced Users sss 16 BioPORTER Reagent 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 protein delivery reagent is the late
18. medium speed 2 2 5 Bring the final volume of the BioPORTER protein mixture to 1000 ul with serum free medium 2 2 6 Aspirate the medium from the cells to be tested wash one time with serum free medium optional and then transfer the BioPORTER protein mixture directly onto the cells 2 2 7 Incubate cells in a 5 CO incubator at 37 C for 4 hours If incubation time needs to be longer than 4 hours add 1000 ul 1 volume of 20 serum containing medium directly to the well BioPORTER Reagent Genlantis a division of Gene Therapy Systems Inc Rev 03152006 l Ph 888 428 0558 e www genlantis com 2 3 Delivery of granzyme B and caspase 3 into Jurkat or Ki Ras 267 p1 cells for 24 well plates 2 3 1 For adherent cells such as Ki Ras 267 B1 prostate cancer cell line seed 0 5 x 10 in 24 well and let grow overnight For Jurkat cells see section 2 3 6 2 3 2 Pipette 2 5 ul of BioPORTER reagent dissolved in chloroform or methanol into the bottom of each eppendorf tube Evaporate the solvent as described above section 2 1 2 2 3 3 Dilute caspase 3 at 330 660 pg ul or granzyme B at 15 45 ng ul in HBS buffer Buffer formula is available in section 1 2 2 2 3 4 Hydrate the BioPORTER reagent dry film with 10 pl of the diluted protein solution Pipette up and down 3 5 times Incubate at room temperature for 3 5 minutes vortex briefly and gently at low medium speed for few seconds 2 3 5 For adherent cells such as Ki Ras 2
19. nants FITC Antibody 1 Analysis by gel electrophoresis and measurement of fluorescence Positive Control 2 Testing for intracellular delivery by the BioPORTER reagent in NIH 3T3 cells B galactosidase 1 Testing for intracellular delivery by the BioPORTER reagent in NIH 3T3 Positive Control cells BioPORTER Reagent Genlantis a division of Gene Therapy Systems Inc Rev 03152006 14 Ph 888 428 0558 e www genlantis com Troubleshooting Guide Problem Possible Causes Recommended Solutions Low delivery Solubilization of BioPORTER Make sure you use chloroform or methanol to solubilize BioPORTER efficiency reagent reagent Vortex vigorously Aliquoting of the BioPORTER Be sure to aliquot the BioPORTER reagent to the bottom of the eppendorf reagent tube Drying of BioPORTER reagent Use sufficient time to air dry or vacuum dry BioPORTER reagent If used inert gas to dry the reagent make sure that BioPORTER reagent did not splash on the side of the tube Amount of BioPORTER Vary the amount of reagent as recommended in the optimization protocol reagent Protein peptide concentration Titrate the concentration and the hydration volume of BIoPORTER reagent Hydration buffers Change the protein dilution buffer and or the pH to improve the delivery Mixing of BioPORTER reagent Allow the mixtures to form for at least 3 minutes Mix well by pipetting up and protein an
20. of protein peptide to be delivered and vary the quantity of BioPORTER reagent see table below The BioPORTER reagent interacts with your molecules of interest via hydrophobic and electrostatic interactions and because each molecule will have different charge and hydrophobicity the amount of BioPORTER reagent may need to be changed Although BioPORTER reagent is not cytotoxic at the recommended concentrations it may show some signs of cytotoxicity at higher reagent to cells concentration ratios Tissue Culture Dish BioPORTER range ul 0 25 1 5 1 25 5 2 5 7 5 5 15 15 30 25 45 96 well 24 well 12 well 6 well 60mm dish 100mm dish After you identify the correct amount of BioPORTER reagent and protein to be used for the delivery reaction you can then optimize the volume used to hydrate the BioPORTER dry film step 1 2 3 To test this parameter fix the protein amount and vary the hydration volume for BioPORTER reagent see Table 4 in section 1 2 3 Different dilution buffers for your protein solution such as Tris HBS and PBS buffers can be tested We have found that for some molecules the buffer composition may be critical Indeed with B galactosidase the efficiency is very good with PBS buffer but not with Tris buffer Whereas with dextran sulfate HBS is the right buffer Also apH may also be critical for some molecules because of their different charge and hydrophobicity varying the pH may help interaction with the BIoPORT
21. ptide to be delivered first and then varying the amount of BioPORTER reagent if necessary Further optimization guidelines are offered in the Appendix section on page 13 Table 2 Suggested Volume of BioPORTER reagent for Various Assays Tissue Culture Dish BioPORTER Number of reactions kit Volume ul 96 well 1 240 24 well 2 5 96 12 well 5 48 6 well 10 24 60mm dish 20 12 100mm dish 35 7 BioPORTER Reagent Genlantis a division of Gene Therapy Systems Inc Rev 03152006 7 Ph 888 428 0558 e www genlantis com 1 1 4 Leave the eppendorf tubes containing the BIoPORTER reagent under a laminar flow hood to evaporate the solvent for at least 2 hours at room temperature For larger volumes evaporate for at least 3 4 hours Alternatively you can use an inert gas like argon or nitrogen to gently and quickly evaporate the solvent by blow drying To avoid splashing the BioPORTER reagent apply the gas flow gently and gradually Make sure that the reagent stays at the bottom of the tube 1 1 5 Optionally you may vacuum the BioPORTER dry film for 1 2 additional hours to completely remove any trace of solvent IMPORTANT The quality of the delivery reaction is severely affected by the presence of methanol or chloroform in the solution Make sure that you follow the 2 4 hours recommendation for drying to assure the removal of all traces of solvent before adding your protein or peptide NOTE We recommend that you aliquot the BioPORTER
22. rst hours of incubation is inhibitory for delivery Make sure that the first 3 4 hours of incubation are done in serum free conditions followed by growth in serum containing medium 1 2 8 1 2 9 Proceed with your experiment for observation or detection assays Cells can be fixed or can be observed alive Two positive controls are provided in the Kit The fluorescein antibody and B galactosidase can be tested as described below in the example protocols 2 Example Protocols 2 1 Delivery of a Fluorescent Antibody p galactosidase or dextran sulfate High and low Molecular Weights for 24 well plates or 22 mm cover slips 2 1 1 BioPORTER Reagent Genlantis a division of Gene Therapy Systems Inc Rev 03152006 Seed 0 5 to 1 x 10 cells per well in a 24 well plate or on a cover slip and let grow overnight Pipette 2 5 ul of BioPORTER reagent dissolved in 250 ul of methanol or chloroform into the bottom of each eppendorf tube Evaporate the solvent under a laminar flow hood for 2 hours or blow dry with an inert gas Optionally vacuum the tube for 2 hours to completely remove any trace amount of solvent Proceed to the next step 2 1 4 or store the tubes at 20 C until use Dilute 0 5 2 ug of FITC Ab dextran sulfate or B galactosidase in 10 to 25 ul of HBS or PBS For f galactosidase we recommend using PBS buffer formulas available in section 1 2 1 The FITC Ab and f galactosidase control proteins provided in
23. sidase caspase 3 caspase 8 and granzyme B have been successfully delivered into the cytoplasm of a variety of different adherent and suspension cells with BioPORTER reagent Furthermore apoptotic proteins such as granzyme B caspase 3 or caspase 8 delivered into cells with the BioPORTER reagent are functional since they can drive cells into apoptosis Now 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 Gene Therapy Systems Inc has extensively tested BioPORTER reagent and verified its effectiveness in delivering active molecules into a wide variety of cells BioPORTER reagent is a new and powerful tool in the functional genomics and proteomics arsenal The BioPORTER reagent is Economical and easy to use Effective in multiple cell types Table 1 Non cytotoxic Fast and efficient Optimum delivery obtained 3 to 4 hours after incubation A non covalent complex Stable with an extended shelf life at 20 C Table 1 Cell Types Successfully Tested HeLa S3 BHK 21 293 CHO K1 NIH 3T3 CV 1 B16 F0 COS 1 K562 COS 7 Jurkat Ki Ras 267 B1 HepG2 MDCK HeLa P19 BioPORTER Reagent Genlantis a division of Gene Therapy Systems Inc Rev 03152006 5 Ph 888 428 0558 e www genlantis com Summary of the BioPORTER Protein Delivery Mechanism
24. st innovation at Gene Therapy Systems Inc 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 reagent is provided in a dried form and needs to be resuspended in methanol or chloroform before use according to section 1 1 on page 7 Catalog Number Description Size or Amount Number of Tubes BP502401 1 BioPORTER Reagent dried 24 reactions 6 well plates or 35 mm dish or 96 reactions 24 well plates 1 B galactosidase control protein 10ug at 100 ug ml 1 FITC antibody control protein 10ug at 100 ug ml fluorescein labeled goat IgG BP509604 4 BioPORTER Reagent dried 4 x 24 Reactions 6 well plates or 35 mm dish or 4 x 96 reactions 24 well plates 1 B galactosidase control protein 10ug at 100 ug ml 1 FITC antibody control protein 10ug 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 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 E mail Orders genlantis com Web Site http www genlantis com Stability and Storage The BioPORTER reagent is shipped frozen Upon receipt and for long term use store all
25. the kit are ready to use without further manipulation Just thaw and mix them well before use Hydrate the BioPORTER reagent dry film with 10 25 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 few seconds Bring the final volume of the BioPORTER protein mixture to 250 ul with serum free medium For 24 well plates aspirate the medium from the cells to be tested wash once with serum free medium optional and then transfer the BioPORTER protein mixture directly onto the cells 10 Ph 888 428 0558 e www genlantis com If cover slips are used blot the cover slip dry and place it in a 35 mm dish Transfer the BioPORTER protein mixture directly onto the cells 2 1 7 Incubate cells in a 5 CO incubator at 37 C for 4 hours Add 250 ul 1 volume of 2096 serum containing medium directly to the 24 well if the incubation time needs to be longer than 4 hours 2 1 8 For cover slips add 1 to 2 ml of growth medium to the 35 mm dish containing the cover slip if the incubation time needs to be longer than 4 hours 2 1 9 After the incubation wash the cells twice with PBS and proceed with the appropriate assay Fluorescent microscopy after washing cells growing on cover slips are mounted directly onto a hanging drop slide with PBS Living cells B galactosidase Assay Sample Protocol are then directly observed
26. up final volume to 250 ul in serum free medium 8 Blot dry coverslips and put in 35 mm dish or for 24 well plates Aspirate old medium 9 Transfer BioPORTER protein medium mixture to cells 10 Incubate cells in a 596 CO incubator at 37 C for 4 hours 11 Add serum containing medium if incubation time needs to be longer than 4 hours 12 After incubation wash cells and proceed with the appropriate assay Delivery of Apoptotic proteins granzyme B caspase 3 or caspase 8 1 Seed 0 5 x 10 adherent cells per well in a 24 well plate and grow O N For suspension cells see step 7 below 2 Pipette 2 5 ul of BIoPORTER dissolved in 250 ul of solvent into an eppendorf tube 3 Evaporate the solvent in a cell culture hood for at least 2 hours Optionally vacuum the BioPORTER reagent film for 2 additional hours 4 Dilute caspase 3 at 330 pg ul 0 1 units ul and granzyme B at 30 ng ul in HBS Use B galactosidase as a negative control by diluting it to 0 1 ug l in PBS 5 Add 10 ul of the diluted protein solution to BIoPORTER reagent dry film and mix by pipetting up and down 3 to 5 times 6 Incubate at room temperature for 3 5 minutes 7 Vortex BioPORTER protein complexes briefly then For adherent cells bring final volume to 200 ul with serum free medium Aspirate the medium from the cells to be tested and then transfer the BioPORTER protein mixture directly onto the cells For suspension cells count and pellet cells resuspend in serum free medium to 0
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