E18 Primary Mouse Cortical Cells, Cat. # N200201 INTRODUCTION
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1. Description Content Amount Day 18 Embryonic C57 Mouse Cortical Cells 1 vial x 2 ml 1 x 108 cells NeuroPure Plating Medium 1 vial x 12 ml NeuroPrep Medium 1 vial x 2 5 ml NeuroPapain Enzyme 1 vial x 5 mg NeuroPure Primary Mouse Cortical Cells are shipped refrigerated Cells are stable for up to 7 days when stored at 4 8 C However WE HIGHLY RECOMMEND PLATING WTHIN 1 TO 2 DAYS FOR BEST RESULTS INTRODUCTION NeuroPure E18 Primary Mouse Cortical Cells Cat N200201 Z Genlantis A division of Gene Therapy Systems Inc Related Products NeuroPure Primary E18 Mouse Hippocampal Cells Amount Catalog 1 x 105 cells N100201 NeuroPure Primary E18 Mouse Hypothalamus Cells 4 Hypothalam N400201 NeuroPure Primary E18 Mouse Striatum Cells 4 Striatum N500201 NeuroPure Primary E18 Mouse Spinal Cord Cells 1 Spinal Cord N600201 NeuroPure Primary E18 Mouse Midbrain Cells 1 Midbrain N700201 NeuroPure Primary E18 Mouse Hippocampal Astrocytes 1 x 105 cells N800201 NeuroPure Primary E18 Mouse Cortical Astrocytes 1 x 10 cells N900201 NeuroPrep Medium 1x 100 ml NM100100 NeuroPapain Enzyme 1x 100 mg NM100200 NeuroFECT Transfection Reagent 75 300 Rxns 7800075 GeneSilencer siRNA Transfection Reagent Cell numbers may vary by or 502. 200 Rxns 7500750 NeuroPure E18 Primary Mouse Cortical Cells are live neurons provided as micro surgically dissected regions of day 18 embryonic C57 mouse brain These cells are prepared fresh each week and shipped in a nutrient rich medium that keeps them alive for up to 14 days under refrigeration Following simple dissociation steps the NeuroPure cells can be quickly plated on almost any poly lysine coated substrate using the provided NeuroPure Plating Medium NeuroPure cells are ideal for a wide variety of applications including transfection pharmacology electrophysiology immunocytochemistry and neuronal development studies MATERIALS AND METHODS Preparation for Culturing 1 Culture plates or coverslips may be prepared by coating with poly D lysine 0 15 ml cm2 50 ug ml 135 kD Sigma P6407 for 1 20 hr Rinse once with 18 Mohm diH20 and let dry Alternatively BD PureCoat poly amine coated culture plates BD Biosciences may be used to reduce formation of neurospheres NOTES If using poly D lysine PDL coated substrate we recommend that you prepare the coated substrate yourself rather than purchasing pre coated PDL substrates e If NeuroPure cells will be cultured for gt 3 4 days we recommend using the culture medium described in Appendix A 2 Clean the Biological Safety Cabinet with 70 alcohol to ensure it is sterile 3 Turn the Biological Safety Cabinet blower on for 10 min before
3. IV Viability Assay Optional 5 Spin the cells at 1 100 rom 200xg for 1 min Discard the Trypan blue provides a rough estimate of cell viability which is supernatant while being careful not to remove any of the cells sufficient for many applications However if a more accurate from the cell pellet quantitation of viability is desired use the following assay 6 Flick the tube a few times to loosen the cell pellet Resuspend 4 Rinse cells twice with PBS the pellet in 1 ml of the provided NeuroPure Plating Medium Resuspend the cells by gently pipetting up and down 7 Aliquot 20 pl and mix with 20 ul of 0 4 trypan blue 2 From an acetone stock of 15 mg ml fluorescein diacetate Sigma add 15 ul 1 100 dilution of the stock into 1 5 ml Hank s Buffered Salt Solution HBSS From an aqueous 8 Count cells with a hemocytometer and determine percentage stock of 4 6 ml ml propidium iodide add 15 ul of the stock into of live cells The expected viability is gt 90 with NeuroPapain the same 1 5 ml HBSS 1 100 dilution Add 40 ul of that treatment and 50 without NeuroPapain treatment dilution to each well with 0 4 ml HBSS further 1 100 dilution 9 Further dilute the cells with NeuroPure Plating Medium to 3 After approximately 1 minute count using Nikon B1A filter or the desired plating density We recommend 32 x 10 cells 2 other blue excitation appropriate for fluorescein fluorescence cm in 0 4 ml 2 cm of substrate Green ce
4. cell culture work 4 Make sure all serological pipettes pipette tips and reagent solutions are sterile 5 Follow the standard sterilization technique and safety rules a Do not pipette with mouth b Always wear gloves and safety glasses when working with animal cells c Handle all cell culture work in a sterile hood ll Enzymatic Pretreatment Optional Enzymatic treatment of NeuroPure tissue prior to mechanical dissociation can increase the number of viable cells by up to 100 However please note that for assays performed within 4 days of plating some digestion of surface proteins is inevitable 1 6 Add 5 mg of NeuroPapain Enzyme into 2 5 ml of NeuroPrep Medium Mix at 37 C for 15 min to completely dissolve the NeuroPapain Sterilize this solution with a 0 2 um filter prior to utilizing for tissue digestion Use within 3 hrs for best results Prior to enzymatic treatment allow the NeuroPure tissue to settle for 15 minutes at 4 C Alternatively place the tube containing the tissue in a 50 ml tube and spin down the cells at 1 100 rpm 200xg for 1 minute Transfer the medium from the NeuroPure cell vial to a separate sterile tube while being careful not to remove any loose tissue pieces Save the medium for trituration following NeuroPapain treatment Immediately add 2 ml of sterile NeuroPapain solution to the tissue containing tube and allow the neuronal tissue to incubate for 30 minutes at 30 C Swirl every 2 m
5. genlantis com Question Answer How many astrocytes do the NeuroPure neuronal After 72 hours of culturing we have measured lt 1 astrocytes However higher cells contain percentages of astrocytes will eventually be generated after several weeks in culture Do you have a detailed protocol for coating of Yes Please see the Hot Files box at the bottom right corner of the NeuroPure web substrates with poly D lysine page in the Cells and Media section of www genlantis com How long does it take for the NeuroPure Cells to Typically the neurites become visible beneath a microscope within 48 72 hours after grow neurites plating Which of your transfection reagents do you For plasmid transfection we recommend the NeuroFECT Transfection Reagent For recommend for transfecting the NeuroPure cells siRNA transfection we recommend our GeneSilencer siRNA Transfection Reagent MV110112 Genlantis Page 2 of 2 10190 Telesis Court San Diego CA 92121 Toll Free 888 428 0558 e U S amp Canada 858 457 1919 e www genlantis com 2010 Genlantis and Gene Therapy Systems Inc
6. in by hand Following incubation spin down the cells at 1 100 rpm 200xg for 1 minute Remove the NeuroPapain solution again being careful not to disturb or remove the tissue Add 1 ml of shipping medium back to the NeuroPure cells Save the other 1 ml of shipping medium for Step 4 below Proceed to Step 3 below Ill Preparation of Isolated Neurons 1 2 After receiving the cells let them settle at 4 C for 15 minutes OR spin down at 1 100 rpm 200xg for 1 min Transfer 1 ml of medium from the cells tube into a sterile 50 ml screw cap tube be careful not to disturb or remove cells from the original cells tube Using a P 1000 pipettor with a sterile blue 1 ml plastic tip 0 8 1 0 mm diameter opening or a silanized 9 inch Pasteur NeuroPure E18 Primary Mouse Cortical Cells pipette with the tip fire polished until it is smooth and has a once the cells adhere 0 8 1 0 mm diameter opening gently pipette the cells with the 10 Incubate the cells at 37 C with 5 CO2 and or 9 or 20 Oz medium up and down into the same container Take care not 11 After 4 days or longer neurons are well differentiated If to create bubbles Repeat this tituration step 15 times or until further culture is desired change half of medium with fresh most all the cells are dispersed warm culture medium See Appendix A 4 Transfer the dispersed cells into the 50ml tube containing the 1 ml of media from Step 2 Gently mix the cells by swirling
7. lls are alive Small red nuclear stain indicates a dead NOTE We do not recommend using antibiotics such as Pen Strep cell If desired fix and stain with 0 25 Coomassie blue R in They have been shown to activate epileptiform bursting ethanol acetic acid H20 45 10 45 1 min rinse with 10 activity in neurons Nevertheless we sometimes start our acetic acid aspirate and dry cultures in gentamicin 10 ug ml and rinse it away after 1 hour APPENDICES Appendix A Culture Medium For culturing NeuroPure cells for gt 3 4 days we recommend the following components from Invitrogen Corporation Neurobasal Medium Cat 21103 B27 Serum Free Supplement Cat 17504 Glutamax Cat 35050 Neurobasal Medium and Glutamax are trademarks of Invitrogen Corporation Appendix B Induction of Neuron Cell Division By addition of Fibroblast Growth Factor 2 Basic FGF2 bFGF at a concentration of 5 ng ml NeuroPure cells will multiply as long as the density is kept below 240 cells mm2 To passage the cells we recommend using NeuroPapain at 2 mg ml in NeuroPrep Medium to incubate the cells for 5 min at 37 C For example a Rinse the cells with warm NeuroPrep Medium b Add 2 mg ml NeuroPapain in NeuroPrep Medium and incubate 5 minutes at 37 C c Carefully remove NeuroPapain and replace with the culture medium from Appendix A Spin to collect cells Appendix C Common Questions Also please see our NeuroPure FAQ s at http www
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