Rat Primary Cortical Astrocytes
Contents
1. 10 11 Remove the spent growth medium from the culture dish containing the cells and store in a sterile tube to use as a washing solution Rinse the surface of the cell layer once with D PBS without Ca and Mg approximately 2 mL D PBS per 10 cm culture surface area by adding the D PBS to the side of the vessel opposite the attached cell layer and rocking back and forth several times Aspirate the D PBS and discard To detach the cells add 3 mL of pre warmed StemPro Accutase Cell Dissociation Reagent per 175 flask adjust volume accordingly for culture dishes of other sizes Incubate for up to 20 minutes at 37 C Rock the cells every 5 minutes and check for cell detachment and dissociation toward single cell under the microscope Once you observe cell detachment gently pipette up and down to break clumps into a single cell suspension Stop the cell dissociation reaction by an adding equal volume of the spent medium from step 1 Disperse the medium by pipetteting over the cell layer surface several times Transfer the cells to a new 15 mL or 50 mL pre rinsed conical tube and centrifuge at 250 x g for 5 minutes at room temperature Aspirate and discard the supernatant Gently resuspend the cell pellet in pre warmed astrocyte growth medium and remove a sample for counting Determine the total number of cells and percent viability using your method of choice If necessary add astrocyte growth medium to th2. Blue Stain 1000 assays L34951 1 unit C10227 Continued on next page Additional Products continued Products for The products listed below may be used for analyzing the Marker phenotype of Rat Primary Cortical Astrocytes In addition to Analysis the primary antibodies listed below Invitrogen offers a variety of isotype specific secondary antibodies conjugated with enzymatic and fluorescent indicators as well as antibody sera and diluents For more information refer to our website www invitrogen com or contact Technical Support see page 13 Item Quantity Cat no Rabbit anti Doublecortin DCX 100 ug 48 1200 Rabbit anti GFAP Glial Fibrillary Acid Protein 1mL 18 0063 DAPI 4 6 diamidino 2 phenylindole dihydrochloride 10 mg D1306 ProLong Gold Antifade Reagent 10 mL P36930 ProLong Gold Antifade Reagent with DAPI 10 mL P36931 vi Introduction Rat Primary Cortical Astrocytes Introduction Source of Rat Primary Cortical Astrocytes Astrocytes are by far the most numerous cell type in the central nervous system CNS outnumbering their neuronal counterparts by approximately tenfold and have critical roles in adult CNS homeostasis Pekny amp Nilsson 2005 They provide biochemical and nutritional support of neurons and endothelial cells which form the blood brain barrier perform the vast majority of synaptic glutamate uptake and maintain extracellula
3. No viable Stock not stored Order new stock and store in liquid cells after thawing stock correctly nitrogen Keep in liquid nitrogen until thawing Cell not handled gently Follow procedures in Thawing Rat Primary Cortical Astrocytes page 5 exactly Fast thawing is the key for a healthy culture Add medium in drop wise manner slowly At time of thawing thaw quickly and do not expose vial to the air but quickly change from nitrogen tank to 37 C water bath Obtain new Rat Primary Cortical Astrocytes Use pre warmed complete growth medium prepared as described on page 4 Generally we recommend a culture density of 2 x 10 cells per cm at the time of recovery Thawing medium not correct Rat Primary Cortical Astrocytes are fragile treat your cells gently do not vortex bang the flasks to dislodge the cells or centrifuge the cells at high speeds Fewer viable cells than expected after thawing stock Cells sticking to plastic culture vessel or pipette tip Prior to use rinse all material that will come in contact with the cells with medium to prevent cells from sticking to the plastic Cells grow slowly or stop growing Growth medium not correct We recommend using astrocyte growth medium for optimal growth and expansion see page 4 Poor serum in growth medium Use Fetal Bovine Serum from a different lot and do not heat inactivate serum Cells have
4. below to grow and maintain Rat Primary Cortical Astrocytes e All solutions and equipment that come in contact with the cells must be sterile Always use proper aseptic technique and work in a laminar flow hood e For consistent results in your experiments we recommend using cells below passage 3 P3 If you expand Rat Primary Cortical Astrocytes beyond P3 we recommend that you perform another round of characterization prior to further experiments e For general maintenance of Rat Primary Cortical Astrocytes as an adherent culture passage cells when they reach 100 confluency at a seeding density of 20 000 cells cm e When thawing or subculturing cells transfer cells into pre warmed medium e Standard physical growth conditions for Rat Primary Cortical Astrocytes are 37 C in a humidified atmosphere of 5 CO in air We recommend that you use Rat Primary Cortical Astrocytes right after recovery After thawing Rat Primary Cortical Astrocytes expand the cells once to have a 1 5 to 2 fold increase in their number and harvest them to use in your experiments e g transplantation experiments metabolic studies Continued on next page Media Requirements Astrocyte Growth Medium You can grow Rat Primary Cortical Astrocytes as an adherent culture on uncoated tissue culture treated vessels For optimal growth and expansion of these cells we recommend using astrocyte growth medium consisting of 85 Dulbecco s
5. day 19 E19 of gestation Cortical e Exhibit gt 70 viability upon thawing Astrocytes Stain 8076 positive for the astrocyte specific marker glial fibrillary acid protein GFAP e Stain lt 10 positive for neuron and oligodendrocyte specific markers galactocerebroside GalC and doublecortin DCX e Exhibit a doubling time of approximately 9 days at P2 e Can be expanded in culture for at least one passage Rat Cortical Primary cells isolated from the cortex of fetal Sprague Dawley Astrocyte rat can be expanded for at least one passage in culture The Culture image below shows Rat Primary Cortical Astrocytes at day five after plating Figure 1 Bright field image of adherent Rat Primary Cortical Astrocytes at passage 2 P2 that have been cultured in astrocyte growth medium for five days The image was captured using 10X objective lens Methods Handling Rat Primary Cortical Astrocytes Guidelines for Culturing Rat Primary Cortical Astrocytes Important As with other mammalian cells when working with Rat Primary Cortical Astrocytes handle as potentially biohazardous material under at least Biosafety Level 1 BL 1 containment For more information on BL 1 guidelines refer to Biosafety in Microbiological and Biomedical Laboratories 5 ed published by the Centers for Disease Control or see the following website www cdc gov od ohs biosfty bmbl5 bmblb5toc htm Follow the general guidelines
6. 063 Neurons DCX 1 400 Rabbit IgG Invitrogen Cat no 48 1200 Oligodendrocytes GalC Millipore Cat no MAB342 1 200 Mouse IgG Continued on next page Characterizing Phenotype of Rat Primary Cortical Astrocytes continued Immunocyto chemistry 10 Fixing Cells 1 Remove culture medium and gently rinse the cells once with D PBS without dislodging Fix the cells with 4 fresh Paraformaldehyde Fixing Solution PFA see Appendix page 13 for recipe at room temperature for 15 minutes Rinse 3X with D PBS containing Ca and Mg Check for presence of cells after fixing Proceed to staining on the next page You may also store slides for up to 3 4 weeks in D PBS at 4 C Do not allow slides to dry Staining Cells 1 Incubate cells for 30 60 minutes in blocking buffer 5 serum of the secondary antibody host species 1 BSA 0 1 Triton X in D PBS with Ca and Mg Note If you are using a surface antigen such as GalC omit Triton X in blocking buffer Remove the blocking buffer and incubate cells overnight at 4 C with primary antibody diluted in D PBS containing 5 serum Ensure that the cell surfaces are covered uniformly with the antibody solution Wash the cells 3X for 5 minutes with D PBS containing Ca and Mg if using a slide use a staining dish with a magnetic stirrer Incubate the cells with fluorescence labeled secondary antibody 5 serum in D PBS
7. Modified Eagle medium containing 4 5 g L glucose and 1576 Fetal Bovine Serum This medium is designed to support isolation and growth of astrocytes derived from cortical tissue of fetal rats e Prepare your growth medium prior to use e When thawing or subculturing cells transfer cells into pre warmed medium at 37 C e We recommend that you aliquot astrocyte growth medium into required working amounts to avoid exposing it to 37 C multiple times Component Cat no Amount Dulbecco s Modified Eagle 11995 065 85 medium high glucose Fetal Bovine Serum 16000 036 15 Thawing Rat Primary Cortical Astrocytes Materials Needed Note Important The following materials are required see page v for ordering information e Rat Primary Cortical Astrocytes stored in liquid nitrogen e Ethanol or 70 isopropanol e Astrocyte growth medium see page 4 pre warmed to 37 C e Disposable sterile 15 mL tubes e Flame polished and autoclaved glass Pasteur pipettes or plastic Pasteur pipettes pre rinsed with growth medium e 37 C water bath e 37 C incubator with a humidified atmosphere of 5 CO e Microcentrifuge e Tissue culture treated flasks plates or Petri dishes uncoated e Hemacytometer cell counter and Trypan Blue LIVE DEAD Cell Vitality Assay Kit or the Countess Automated Cell Counter The Countess Automated Cell Counter is a benchtop instrument designed to measur
8. been passaged too many times Obtain new P1 Rat Primary Cortical Astrocytes 12 Appendix Recipes Para To prepare 20 paraformaldehyde PFA stock solution formaldehyde 1 Add PBS to 20 g of EM grade paraformaldehyde Solution Electron Microscopy Services Cat no 19208 and bring the volume up to 100 mL 2 Add 0 25 mL of 10 N NaOH and heat at 60 C using a magnetic stirrer until completely dissolved 3 Filter through 0 22 micron filter and cool on ice Make sure the pH is 7 5 8 0 4 Aliquot 2 mL in 15 mL tubes freeze on dry ice and store at 20 C To prepare 4 PFA for fixing 1 Add 8 mL PBS into each 15 mL tube containing 2 mL of 2096 PFA and thaw in a 37 C water bath 2 Once dissolved cool on ice 13 Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete Technical Support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Busin
9. cco s Modified Eagle medium containing 4 5 g L glucose and 15 Fetal Bovine Serum plus 10 DMSO Handle cells as potentially biohazardous material under at least Biosafety Level 1 BL 1 containment This product contains Dimethyl Sulfoxide DMSO a hazardous material Review the Material Safety Data Sheet MSDS before handling Material Safety Data Sheets MSDSs are available on our website at www invitrogen com msds Additional Products Additional The products listed in this section may be used with Rat Products Primary Cortical Astrocytes For more information refer to our website www invitrogen com or contact Technical Support see page 13 Item Quantity Cat no Dulbecco s Modified Eagle Medium D MEM 1X 500 mL 11995 065 liquid high glucose 1000 mL 11995 040 Fetal Bovine Serum FBS Certified 100 mL 16000 036 Dulbecco s Phosphate Buffered Saline D PBS 500 mL 14190 144 containing no calcium magnesium or phenol red Dulbecco s Phosphate Buffered Saline D PBS 500 mL 14040 133 containing calcium and magnesium but no phenol red StemPro Accutase Cell Dissociation Reagent 100 mL A11105 01 Trypan Blue Stain 100 mL 15250 061 Trypan Blue Stain 0 4 for use with the Countess Automated Cell Counter 2x1mL T10282 LIVE DEAD Cell Vitality Assay Kit TM Countess Automated Cell Counter includes 50 Countess cell counting chamber slides and 2 mL of Trypan
10. ch cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Life Technologies is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing invitrogen com References Lepore A C Rauck B Dejea C Pardo A C Rao M S Rothstein J D and Maragakis N J 2008 Focal transplantation based astrocyte replacement is neuroprotective in a model of motor neuron disease Nat Neurosci 11 1294 1301 Maragakis N J and Rothstein J D 2006 Mechanisms of Disease astrocytes in neurodegenerative disease Nat Clin Pract Neurol 2 679 689 Pekny M and Nilsson M 2005 Astrocyte activation and reactive gliosis Glia 50 427 434 Rothstein J D Dykes Hoberg M Pardo C A Bristol L A Jin L Kuncl R W Kanai Y Hediger M A Wang Y Schielke J P and Welty D F 1996 Knockout of glutamate transpo
11. e cell count and viability live dead and total cells accurately and precisely in less than a minute per sample using the standard Trypan Blue uptake technique see page v for ordering information Using the same amount of sample that you currently use with the hemocytometer the Countess Automated Cell Counter takes less than a minute per sample for a typical cell count and is compatible with a wide variety of eukaryotic cells Rat Primary Cortical Astrocytes readily stick to the plastic used in cell culture dishes and centrifuge tubes Prior to use rinse all material that will come in contact with the cells with medium to prevent cells from sticking to the plastic To thaw and establish Rat Primary Cortical Astrocytes follow the procedure on the next page Continued on next page Thawing Rat Primary Cortical Astrocytes continued Thawing 1 Remove the cells from liquid nitrogen storage and Procedure immediately transfer the cells to a 37 C water bath to prevent crystal formation 2 Quickly thaw the vial of cells by gently swirling it in the 37 C water bath and removing it when the last bit of ice has melted typically 2 minutes Do not submerge the vial completely Do not thaw the cells for longer than 2 minutes Do not introduce bubbles into the cell suspension as it decreases cell viability 3 When thawed transfer the tube containing the cells into the laminar flow hood and wash the outside of the tube
12. e cells to achieve the desired cell concentration and recount the cells Plate cells in an uncoated tissue culture treated flask plate or Petri dish at a seeding density of 2 x 10 cells per cm Incubate cells at 37 C 5 CO and 90 humidity and change growth medium every 4 5 days Characterizing Phenotype of Rat Primary Cortical Astrocytes Phenotypic Markers Primary Antibodies Immunocytochemical analysis of Rat Primary Cortical Astrocytes using fluorochrome conjugated antibodies to astrocyte specific marker GFAP should indicate 80 expression while the expression of the neuron specific marker DCX and the oligodendrocyte specific marker GalC should be lt 10 See Figure 2 on page 11 for an example of phenotypic marker expression of Rat Primary Cortical Astrocytes cultured in astrocyte growth medium and analyzed using the protocol on the next page The following table lists the primary antibodies used for classifying Rat Primary Cortical Astrocytes See page vi for ordering information Note The behavior of the antibodies and their dilution ratio is dependent on their source and concentration We recommend that you optimize the parameters of your immunocytochemistry experiments e g dilution ratio incubation time if you use antibodies from a source other than listed below Cell Type Antigen Dilution Antibody ratio type Astrocytes GFAP 1 200 Rabbit IgG Invitrogen Cat no 18 0
13. emic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research For products that are subject to multiple limited use label licenses the terms of the most restrictive limited use label license shall control Life Technologies Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Life Technologies Corporation whi
14. ess Park Carlsbad CA 92008 USA 3 9 15 Kaigan Minato ku 3 Fountain Drive Tel 1 760 603 7200 Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail E mail E mail tech supportGinvitrogen com jpinfoGinvitrogen com eurotech invitrogen com Material Safety Data Sheets MSDSs Certificate of Analysis 14 MSDSs Material Safety Data Sheets are available on our website at www invitrogen com msds The Certificate of Analysis provides detailed quality control information for each product Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box Purchaser Notification Limited Warranty Invitrogen a part of Life Technologies Corporation is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This war
15. invitrogen Rat Primary Cortical Astrocytes Catalog no N7745 100 Rev date 14 May 2009 Manual part no A11231 MAN0001664 Contents Goritents and Storage erected tette dite ite tne ne iv Additional Products eto er e aieo v Introduction sinauni sasni onnaa ai inada aaien aina 1 Rat Primary Cortical Astrocytes seen ee nene 1 gp c sete 3 Handling Rat Primary Cortical Astrocytes sse 3 Media Requirements enne nnne nennen 4 Thawing Rat Primary Cortical Astrocytes srrnnevrvrrrerrrrererrarersrserrrsssersssernssnne 5 Expanding Rat Primary Cortical Astrocytes sss 7 Characterizing Phenotype of Rat Primary Cortical Astrocytes 9 Phenotype Marker Expression of Rat Primary Cortical Astrocytes 11 Troubleshooting eet EA A eie rere Red 12 Appendix csi ao ere cee ae rd nu eins testede tese 13 Recipe Sie a asi eta pei EN 13 Technical Support entente ettet teen ien 14 Purchaser Notification inerenti Rete rei insisi 15 References ecce ite cti epe et c ted bet ea bd bee ie pra S 17 Contents and Storage Shipping and Storage Contents Rat Primary Cortical Astrocytes are shipped on dry ice Upon receipt store the cells in liquid nitrogen Amount supplied One vial containing 1 x 10 viable cells Composition 1 mL of cells in freezing medium Freezing medium 90 Astrocyte growth medium 85 Dulbe
16. r potassium levels Rothstein et al 1996 Rothstein et al 1994 Astroglial dysfunction has been implicated in a number of CNS pathologies including amyotrophic lateral sclerosis ALS and ischemic neuronal death Maragakis amp Rothstein 2006 Takano et al 2009 and transplantation based astrocyte replacement therapy has been shown to be a promising therapeutic strategy against neuronal death Lepore et al 2008 Although there are few known differences between cortical and hippocampal astrocytes it has been reported that astrocytes from different regions of the brain show a differential sensitivity to ischemic injury Xu et al 2001 Zhao amp Flavin 2000 Rat Primary Cortical Astrocytes are isolated from the cortices of fetal Sprague Dawley rats at embryonic day 19 E19 of gestation The cells are isolated from tissue under sterile conditions placed through one round of enzymatic dissociation and expansion in astrocyte growth medium 85 Dulbecco s Modified Eagle medium containing 4 5 g L glucose and 15 Fetal Bovine Serum The cells are cryopreserved at passage 1 P1 in 90 astrocyte growth medium plus 10 DMSO Each vial of Rat Primary Cortical Astrocytes contains 1 x 106 cells mL that can be expanded in culture for at least one passage Continued on next page Rat Primary Cortical Astrocytes continued Characteristics Isolated from the brain cortex of fetal Sprague Dawley of Rat Primary rats at embryonic
17. ranty limits the Company s liability to only the price of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 15 Purchaser Notification continued Limited Use Label License No 5 Invitrogen Technology 16 The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an acad
18. rters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate Neuron 16 675 686 Rothstein J D Martin L Levey A I Dykes Hoberg M Jin L Wu D Nash N and Kuncl R W 1994 Localization of neuronal and glial glutamate transporters Neuron 13 713 725 Takano T Oberheim N Cotrina M L and Nedergaard M 2009 Astrocytes and ischemic injury Stroke 40 S8 12 Xu L Sapolsky R M and Giffard R G 2001 Differential sensitivity of murine astrocytes and neurons from different brain regions to injury Exp Neurol 169 416 424 Zhao G and Flavin M P 2000 Differential sensitivity of rat hippocampal and cortical astrocytes to oxygen glucose deprivation injury Neurosci Lett 285 177 180 2009 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 17 invitrogen Corporate Headquarters Invitrogen Corporation 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
19. with 70 isopropanol 4 Very gently transfer the cells into a pre rinsed 15 mL centrifuge tube using a Flame polished and autoclaved glass Pasteur pipette or a pre rinsed plastic Pasteur pipette 5 Rinse the vial with 1 mL of astrocyte growth medium and dropwise add to the cells in the 15 mL centrifuge tube one drop second Mix by gentle swirling after each drop 6 Dropwise add 8 mL of astrocyte growth medium to the cell solution and mix gently Centrifuge the cells at 250 x g for 5 minutes Aspirate the supernatant and resuspend cells in 2 mL of astrocyte growth medium 9 Determine the viable cell count using your method of choice 10 Plate the cells at a seeding density of 2 x 10 cells per cm on an uncoated tissue culture treated culture dish If necessary gently add growth medium to the cells to achieve the desired cell concentration and recount the cells 11 Incubate at 37 C 5 CO and 90 humidity Replace the medium with an equal volume of fresh pre warmed astrocyte growth medium every 4 5 days 12 Passage cells when the culture is 100 confluent Expanding Rat Primary Cortical Astrocytes Introduction Materials Needed Important You may expand Rat Primary Cortical Astrocytes as an adherent culture on uncoated tissue culture treated flasks plates or dishes Subculture your cells when 100 confluent Note We recommend that you use Rat Primary Cortical Astrocytes right after recover
20. with Ca and Mg in the dark at 37 C for 30 45 minutes Wash the cells 3X with D PBS containing Ca and Mg and in the last wash counter stain with DAPI solution 3 ng mL for 5 minutes and rinse with D PBS If desired mount with 3 drops of ProLong Gold antifade reagent per slide and seal with the cover slip see page vi for ordering information You may store the slides in the dark at 4 C Phenotype Marker Expression of Rat Primary Cortical Astrocytes Astrocyte The image below shows the phenotype marker expression of specific Rat Primary Cortical Astrocytes cultured in astrocyte growth Marker medium for eleven days and analyzed using the protocol on Expression the previous page Figure 2 Rat Primary Cortical Astrocytes stained by indirect immunofluorescence for the intracellular marker GFAP red Nuclei were stained with DAPI blue The cells were maintained in astrocyte growth medium 85 Dulbecco s Modified Eagle medium containing 4 5 g L glucose and 15 Fetal Bovine Serum for eleven days prior to 4 paraformaldehyde fixation and staining While 80 of the cells stain positive for GFAP lt 10 of the cells show neuron specific DCX and oligodendrocyte specific GalC expression data not shown Scale bar 200 um 11 Troubleshooting Culturing The table below lists some potential problems and solutions that Cells help you troubleshoot your cell culture problems Problem Cause Solution
21. y After thawing Rat Primary Cortical Astrocytes expand the cells once to have a 1 5 to 2 fold increase in their number and harvest them to use in your experiments e g transplantation experiments metabolic studies The following materials are required for passaging Rat Primary Cortical Astrocytes see page v for ordering information e Culture vessels containing Rat Primary Cortical Astrocytes 100 confluent e Uncoated tissue culture treated flasks plates or Petri dishes e Astrocyte growth medium see page 4 pre warmed to 37 C e Disposable sterile 15 mL or 50 mL conical tubes pre rinsed with medium e 37 C incubator with humidified atmosphere of 5 CO e Dulbecco s Phosphate Buffered Saline D PBS containing no calcium magnesium or phenol red e StemPro Accutase Cell Dissociation Reagent see page v pre warmed to 37 C e Hemacytometer cell counter and Trypan Blue LIVE DEAD Cell Vitality Assay Kit or the Countess Automated Cell Counter Rat Primary Cortical Astrocytes readily stick to the plastic used in cell culture dishes and centrifuge tubes Prior to use rinse all material that will come in contact with the cells with medium to prevent cells from sticking to the plastic To passage Rat Primary Cortical Astrocytes follow the procedure on the next page Continued on next page Expanding Rat Primary Cortical Astrocytes continued Passaging Rat Primary Cortical Astrocytes
Download Pdf Manuals
Related Search