Neuronal Transfection Reagent
Contents
1. NeuroF ECT a Neuronal Transfection Reagent A division of Gene Therapy Systems Inc Contents Related Products Continued Catalog Content Amount Catalog Content Amount T800075 NeuroFECT Transfection Reagent 75 300 rxns 10 75 ml N300200 NeuroPure P8 Rat Cerebellar Cells 4 x10 cells T800750 NeuroFECT Transfection Reagent 375 1500 rxns 5 x 0 75 ml N400200 NeuroPure E18 Primary Rat Hypothalamus 1 pair N500200 NeuroPure E18 Primary Rat Striata 1 pair Related Products N600200 NeuroPure E18 Primary Rat Spinal Cord 1 spinal cord Catalog Content Amount N700200 NeuroPure E18 Primary Rat Midbrain 1 midbrain N100200 NeuroPure E18 Primary Rat Hippocampal Cells 1 x 10 cells N100300 NeuroStem E18 Rat Hippocampal Progenitors 1 x 108 cells N200200 NeuroPure E18 Primary Rat Cortical Cells 2 x 10 cells N200300 NeuroPure E18 Rat Cortical Progenitors 2 x 108 cells Shipping and NeuroFECT Transfection Reagent is shipped at room temperature For maximum stability store all reagents at 4 C upon receipt Storage When stored properly the NeuroFECT reagent is stable for 6 months INTRODUCTION NeuroFect is a novel biodegradable cationic polymer created specifically for optimal transfection of neuronal cells During transfection the polymer DNA complexes polyplexes are endocytosed into the cells where the polymer is biodeg2. E 60 mm 6 8 ug 1ml 2 5 ml 100 mm 8 12 ug 2 ml 5 ml SFM Serum Free Medium Dilute the NeuroFect amount inidcated in half this volume and the DNA amount in the other half of this volume Volume prior to adding NeuroFECT DNA Complexes 13 Optimization of cell plating densities for transfection of primary cells should start using densities giving maximum cell health during routine culturing Greater or lessor densities can then be tested Optimization of plating densities for transfection of neuronal cell lines should start at those giving 50 70 confluency on the day of transfection 14 For addressing any cytotoxicity that may occur it is recommended to reduce the amount of NeuroFECT reagent used in 15 increments until the cytotoxicity is eliminated License The purchase price paid for the NeuroFect Transfection Reagent Kit grants end users a non transferable non exclusive license to use the kit and or its components for internal research use only as described in this manual in particular research use only excludes and without limitation resale repackaging or use for the making or selling of any commercial product or service without the written approval of Genlantis Separate licenses are available for non research use or applications The NeuroFect Transfection Reagent Kit is not to be used for human diagnostic or included used in any drug intended for human use Care and attention should be exercised in handling the
3. kit components by following appropriate research lab practices Purchasers may refuse this license by returning the enclosed materials unused By keeping or using the enclosed materials you agree to be bound by the terms of this license The laws of the State of California shall aovern the interoretation and enforcement of the terms of this Licence Version MV090106 Genlantis Page 2 of 2 Phone 858 457 1919 888 428 0558 U S Toll free e Fax 858 623 9494 www genlantis com
4. obasal B27 0 5 mM glutamine Add 0 25 uM glutamate for hippocampal Primary Neuron Type 24 well Plate 96 Well Plate neurons Cortical Neurons 80 000 cells well 20 000 cells well ory 0 co Ninel eee ave eR Sie COR See Hippocampal Neurons 65 000 cells well 15 000 cells well 3 After 3 days remove half of the plating medium volume per well and replace with same amount of the primary Media Volume 500 pl well 100 pl well neuronal culture medium indicated in step 1 above Do not use glutamate at this point for hippocampal neurons Table 1 Cell Densities and Media Volume per Well 4 Continue culturing the cells for an additional 3 4 days Version MV090106 Genlantis Page 1 of 2 Phone 858 457 1919 888 428 0558 U S Toll free e Fax 858 623 9494 www genlantis com C Transfection 5 Prepare the NeuroFECT and DNA in separate tubes according to Table 2 Dilute the NeuroFECT in half the Serum Free Medium SFM volume indicated in Table 2 and your DNA in the other half of SFM e g for Primary Hippocampal Neurons in 24 well plates dilute 4 ug of NeuroFECT in 50 ul of SFM and 1 0 ug in 50 pl of SFM Table 2 NeuroFECT DNA and SFM Amounts for Complex Formation in Primary Rat Hippocampal and Cortical Neurons 24 well plates amounts per well 96 well plates amounts per well Neuron Type Reagent Amounts Reagent Amounts NeuroFECT DNA SFM NeuroFECT DNA SFM Primary Hippocampal Neuron
5. raded into small non toxic molecules The ability of NeuroFect to biodegrade in vivo dramatically reduces its cytotoxicity and therefore maximizes the delivery of macromolecules into cells NeuroFect is compatible with serum containing media is easy to use and provides the highest possible transfection efficiencies for your primary neurons MATERIALS AND METHODS A Preliminary Notes The NeuroFECT Reagent is provided at a stock concentration of 5 yg ul You may use the reagent at the stock concentration or dilute it to 1 ug ul for easier pipetting and transfection optimization Use only sterile water or serum free medium such as OptiMem Medium Invitrogen Corporation Cat 31985 070 for dilution If diluting with serum free medium NeuroFECT should be used within 30 minutes Il The following protocol was derived from optimizing transfection of Primary E18 Rat Hippocampal Neurons Cat N100200 and Primary E18 Rat Cortical Neurons Cat N200200 The DNA amounts NeuroFECT DNA ratios cell plating densities and timing of transfection may vary for other types of primary neurons and neuronal cell lines However the values given below for these parameters should function as good starting points Optimization guidelines are provided on Page 2 B Preparation and Growth of Primary Neurons 1 Seed neurons on freshly coated poly lysine coated plates in the densities indicated in Table 1 using the following media for primary neurons Neur
6. s 4 ug 1 0 ug 100 ul 1 5 ug 0 25 ug 50 ul Primary Cortical Neurons 5 ug 1 0 ug 100 ul 1 75 ug 0 25 yg 50 pl SFM Serum Free Medium Dilute the NeuroFect amount inidcated in half this volume and the DNA amount in the other half of this volume 6 For NeuroFECT DNA Complex Formation add the reagents in this order diluted NeuroFECT to diluted DNA in a drop wise fashion and mix with gentle pipetting 7 Incubate the NeuroFECT DNA complex for 15 20 minutes at room temperature 8 Remove half the old culture medium from the cells and replace with half the volume of fresh culture medium indicated in Table 1 9 Add the NeuroFECT DNA complexes to the cells Gently mix by swirling plate 10 Incubate the cells at 37 C in 5 CO2 11 Perform gene expression assay 24 48 hours later D Transfection Optimization Guidelines 12 To obtain the maximum transfection efficiency for other cell types besides primary cortical and hippocampal neurons and for other tissue culture dish sizes besides 24 well and 96 well plates we recommend using the above mentioned DNA quantities and NeuroFECT DNA ratios as starting points and then testing different conditions according to the following guidelines Table 3 Transfection Optimization Guidelines Tissue Culture Dish NeuroFECT DNA Ratio DNA SFM Media Volume 96 well 0 1 0 5 ug 50 ul 100 ul 24 well 0 5 2 ug 100 ul 500 ul 6 well PAED 2 6 500 ul 1ml ug p9
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