E18 Primary Mouse Hippocampal Cells, Cat. # N800201
Contents
1. Amount 1 vial x 2 ml Description Content Day 18 Embryonic C57 Mouse Hippocampal Astrocytes 1 x 105 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 Hippocampal Astrocytes 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 NeuroPure E18 Primary Mouse Hippocampal Cells Cat N800201 Z Genlantis A division of Gene Therapy Systems Inc Related Products NeuroPure Primary E18 Mouse Hippocampal Cells 1 x 105 cells Amount Catalog N100201 NeuroPure Primary E18 Mouse Cortical Cells 1 x 108 cells N200201 NeuroPure Primary E18 Mouse Hypothalamus Cells 1 Hypothalam N400201 NeuroPure Primary E18 Mouse Striatum Cells 1 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 Cortical Astrocytes 1 x 106 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 200 Rxns 7500750 INTRODUCTION2. Pellet the cells as in Step Ill 1 and continue with Steps III 2 IlI 10 IV Viability Assay Optional Trypan blue provides a rough estimate of cell viability which is sufficient for many applications However if a more accurate quantitation of viability is desired use the following assay Rinse cells twice with PBS From an acetone stock of 15 mg ml fluorescein diacetate Sigma add 15 pl 1 100 dilution of the stock into 1 5 ml Hank s Buffered Salt Solution HBSS From an aqueous stock of 4 6 ml ml propidium iodide add 15 ul of the stock into the same 1 5 ml HBSS 1 100 dilution Add 40 ul of that dilution to each well with 0 4 ml HBSS further 1 100 dilution After approximately 1 minute count using Nikon B1A filter or other blue excitation appropriate for fluorescein fluorescence Green cells are alive Small red nuclear stain indicates a dead cell If desired fix and stain with 0 25 Coomassie blue R in ethanol acetic acid H20 45 10 45 1 min rinse with 10 acetic acid aspirate and dry Appendix B Common Questions Also please see our NeuroPure FAQ s at http www genlantis com Question Answer Do you have a detailed protocol for coating of substrates with poly D lysine Yes Please see the Hot Files box at the bottom right corner of the NeuroPure web page in the Cells and Media section of www genlantis com What concentration of B27 Supplement should be used with the Neurobasal medium
3. 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 ie ale poy ey oe pr a ate a ae the NeuroPure cell vial to a separate sterile tube while being al VOU ee 1G ee ee eee greg eer Man careful not to remove any loose tissue pieces Save the parehasing pre coated jes SUUSTAAN medium for trituration following NeuroPapain treatment e If NeuroPure cells will be cultur ed for gt 3 4 days W 3 Immediately add 2 ml of sterile NeuroPapain solution to the recommend using the culture medium described in Appendix A tissue containing tube and allow the neuronal tissue to 2 Clean the Biological Safety Cabinet with 70 alcohol to incubate for 30 minutes at 30 C Swirl every 2 min by hand ensure it is sterile 4 Following incubation spin down the cells at 1 100 rpm 200xg 3 Turn the Biological Safety Cabinet blower on for 10 min for 1 minute Remove the NeuroPapain solution again being before cell culture work careful not to disturb or remove the tissue 4 Make sure all serological pipettes pipette tips and reagent 5 Add 1 ml of shipping medium back to the NeuroPure cells solutions are sterile Save the other 1 ml of shipping medium for Step 4 below 5 Follow the standard sterilization technique and safety rules 6 Proceed to Step 3 below a Do not pipette with mouth b Always wear gloves and safety glasses when working with animal cells Ill Preparation of Isolat
4. Cell numbers may vary by or 50 NeuroPure E18 Primary Mouse Hippocampal Astrocytes are live cells 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 plating some digestion of surface proteins is inevitable 1 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 Alternatively BD PureCoat poly amine coated culture plates 2 Prior to enzymatic treatment allow the NeuroPure tissue to BD Biosciences may be used to reduce formation of settle for 15 minutes at 4 C Alternatively place the tube neurospheres
5. The B27 Supplement is provided at a 50x concentration It should be diluted to a 1x concentration in the Neurobasal medium How long does it take for the NeuroPure Cells to grow neurites plating Typically the neurites become visible beneath a microscope within 48 72 hours after Which of your transfection reagents do you recommend for transfecting the NeuroPure cells For plasmid transfection we recommend the NeuroFECT Transfection Reagent For 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. ed Astrocytes 1 After receiving the cells let them settle at 4 C for 15 minutes c Handle all cell culture work in a sterile hood OR spin down at 1 100 rpm 200xq for 1 min 2 Transfer 1 ml of medium from the cells tube into a sterile 50 ll Enzymatic Pretreatment Optional ml screw cap tube be careful not to disturb or remove cells Enzymatic treatment of NeuroPure tissue prior to mechanical from the original cells tube dissociation can increase the number of viable cells by up to 100 3 Using a P 1000 pipettor with a sterile blue 1 ml plastic tip 0 8 However please note that for assays performed within 4 days of 1 0 mm diameter opening or a silanized 9 inch Pasteur NOTE NeuroPure E18 Primary Mouse Hippocampal Astrocytes pipette with the tip fire polished until it is smooth and has a 0 8 1 0 mm diameter opening gently pipette the cells with the medium up and down into the same container Take care not to create bubbles Repeat this tituration step 15 times or until most all the cells are dispersed Transfer the dispersed cells into the 50ml tube containing the 1 ml of media from Step 2 Gently mix the cells by swirling Spin the cells at 1 100 rpm 200xg for 1 min Discard the supernatant while being careful not to remove any of the cells from the cell pellet Flick the tube a few times to loosen the cell pellet Resuspend the pellet in 1 ml of the provided NeuroPure Plating Medium Resuspend the cells by gent
7. ly pipetting up and down Aliquot 20 yl and mix with 20 ul of 0 4 trypan blue Count cells with a hemocytometer and determine percentage of live cells The expected viability is gt 90 with NeuroPapain treatment and 50 without NeuroPapain treatment Further dilute the cells with NeuroPure Plating Medium to the desired plating density We recommend 32 x 10 cells 2 cm in 0 4 ml 2 cm of substrate We do not recommend using antibiotics such as Pen Strep They have been shown to activate epileptiform bursting activity in neurons Nevertheless we sometimes start our cultures in gentamicin 10 ug ml and rinse it away after 1 hour once the cells adhere Incubate the cells at 37 C with 5 CO2 and or 9 or 20 Oz 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 11 After 4 days or longer astrocytes will be nearly confluent and ready to harvest or pass If desired you can change 50 of the medium to NeuroBasal B27 0 5 mM Glutamax 2 Horse Serum See Appendix in preparation to harvest the next day cultures If expansion is desired harvest cells with 0 05 Trypsin Cat NM100200 in NeuroPrep Media Cat NM100100 at 37 for 5 minutes
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