User Manual - Cyagen Biosciences
Contents
1. GXXAC 90011 Fedoroff G and Richardson A 1992 Protocols for Neural Cell Cultures Humana Press Totowa N J Jeffrey W Allen Lysette A Mutkus and Michael Aschner 2001 Isolation of Neonatal Rat Cortical Astrocytes for Primary Cultures Current Protocols in Toxicology 12 Cyagen Biosciences reserves all rights on the technical documents of its OriCell cell culture products No part of this document may be reproduced or adapted for other purposes without written permission from Cyagen Biosciences IMPIOO44A2 FCCAC 00001 Page 8 of 82. ae Cyd J C n We help gou discover life OriCell Fisher 344 F344 Rat CorticalAstrocytes Cat No FCCAC 00001 cp cyagen Table of Contents CONLENES and Storag Cvssanicvesspesesnatsciesceanccacavaissnecsanseiuneenasanansavuaiannccieneasnanisamenaaciuuenanpenianae 3 Product Introductio Merissa a E 3 Product ADDIICALION S iinei E TA AAEN A General Handling PrinCGl PleS visciiscsdracsvacensiessarsanaranannissniuasenssseseneeantnuieensansecnaseienedeeseanannnenti 4 Culture OriCell F344 Rat Cortical Astrocytes Thawing and Establishing OriCell F344 Rat Cortical AStrocyteS sssssssssssssnnunnnn aes 4 Passaging of OriCell F344 Rat Cortical AStrocyteS cccscccccceccecucuseecueeneeeueeeeueueerueeneueenes 6 Cryopreservation of OriCell F344 Rat Cortical AStroCyteS sssssssssssnnnnnnnnnnnnnnnnnnnnnnn 7 APPEN eT E EA Related PROGUCUS se sinccuceiesccevew seuss sccseesuceadiecreunssecesesk cece NANNAN 8 ELALLA SS A E E EEEE EEE EE 8 Tecnhical SUPPOFE svriniriinsrika areni co cyagen CONTENTS AND STORAGE Product Name Fisher 344Rat Cortical Astrocytes Catalog No FCCAC 00001 Amount per Vial 1x10 Cells Cryopreserved At First Passage Storage Condition Liquid Nitrogen CAUTION Please handle this product as a potentially biohazardous material This A product contains Dimethyl Sulfoxide DMSO a hazardous material in the freezing medium PRODUCT INTRODUCTION Astrocytes are characteristic star shaped g
3. e 3 0 4 0x10 cells cm 3 For all studies it is strongly recommended to use cells that are at or under an original passage number of 5 4 For general maintenance of cells we recommend that the medium is changed if it becomes acidic the pH indicator in medium appears yellow In general change the growth medium every three days 5 Do not let OriCell F344 Rat Astrocyte overgrow as it will result in contact inhibition When the cells are 80 90 confluent subculturing the cells is strongly recommended Note We strongly recommend the use of OriCell culture media and other related reagents for optimal results THAWING AND ESTABLISHINGOriCell Fisher 344 F344 RAT CORTICAL ASTROCYTES N Materials Required e OriCell Astrocyte Growth Medium Cat No GXXAC 90011 Thawing and EstablishingF344RatCortical Astrocytes Pre warm OriCell Astrocyte Growth Medium to 37 C Add 9mIL of OriCell Astrocyte Growth Medium to a 15mL conical tube Remove the cryovial of OriCell F344 Rat Cortical Astrocytes from liquid nitrogen ee ee ee Quickly thaw the vial in a 37 C water bath until the last ice crystal disappears and finish the thawing procedure within 3 minutes Be careful not to submerge the entire vial Maximum cell viability is dependent on the rapid and complete thawing of frozen cells Note Results will be less than optimal if the cells are thawed for more than 3 minutes IMPIOO44A2 FCCAC 00001 Pag
4. e 4 of 8 O cyagen 5 As soon as the cells are completely thawed disinfect the outside of the vial with 70 v v ethanol 6 Use a pipette to transfer the cells to the 15mL conical tube containing OriCell Astrocyte Growth Medium inside a biosafety cabinet Be careful not to introduce any bubbles during the transfer process 7 Rinse the vial with 1mML of medium to reduce the loss of cell and then transfer this 1mL of cell suspension to the conical tube 8 Gently mix the cell suspension by slowly pipetting up and down Be careful not to introduce any bubbles 9 Centrifuge the cell suspension at 250 x g for 5 minutes 10 Carefully aspirate off as much of the supernatant as possible and add 2 3mL of fresh OriCell Astrocyte Growth Medium pre warmed to 37 C 11 Gently re suspend the cells in OriCell Astrocyte Growth Medium 12 Seed the cells into a T25 flask and add a sufficient OriCell Astrocyte Growth Medium Gently rock the culture flask to evenly distribute the cells 13 Incubate the flask at 37 C in a 5 CO humidified incubator 14 The next day change the medium with fresh OriCell pre warmed to 37 C Astrocyte Growth Medium 15 Change the growth medium every three days thereafter 16 When the cells are approximately 80 90 confluent they can be dissociated with Trypsin EDTAand passaged Note Changing Medium 1 Warm an appropriate amount of medium to 37 C in a sterile container Rem
5. imately80 are rounded up At this point gently tap the side of the flask to release themajority of cells from the culture surface Note Avoid leaving cells exposed to the trypsin longer than necessary Care should also be taken that the cells are not forced to detach prematurely as this may result In clumping 7 After the cells are visibly detached immediately add the pre warmed OriCell Astrocyte Growth Medium 6mL for T75 flask 3mL for T25 flask to neutralize the trypsinization 8 Gently pipet the medium over the cells to dislodge and resuspend the cells Repeat 5 6 times until all the cells are dissociated from the flask and evenly dispersed into a single cell suspension Note Care should be taken to avoid introducing bubble during pipetting 9 Transfer the dissociated cells into a 15mL conical tube 10 Centrifuge at 250 x g for 5 minutes to pellet the cells 11 Carefully aspirate off as much of the supernatant as possible IMPIOO44A2 FCCAC 00001 Page 6 of 8 co cyagen 12 Add 2ml of OriCell Astrocyte Growth Medium to the conical tube and gently re Suspend the cells thoroughly 13 Plate the cells into appropriate flasks OriCell F344Rat Cortical Astrocytes can be splitat 1 2 or other appropriate ratio 14 Add an appropriate amount of medium to the cells Incubate the cells at 37 C in a 5 CO humidified incubator Additional Tips Time to Change Medium It is recommended tochange the cultu
6. lial cells in the brain and spinal cord They are crucial in forming the blood brain barrier through biochemical support of endothelial cells providing nutrients to the nervous tissue maintaining of extracellular ion balance and healing of the brain and spinal cord from traumatic injuries OriCell Fisher 344 F344 Rat Cortical Astrocytes are derived from the cortex of newborn F344rats They are tested negative for bacteria fungi and mycoplasma Activity of CyagenOriCell F344 Rat Cortical Astrocytes e Recovery Viability 280 e Can be passaged at least 2 times Purity of CyagenOriCell F344 Rat Cortical Astrocytes e GFAP positive cells gt 80 e tubulin IIIpositive cells lt 10 e GalCpositive cells lt 10 This product is intended for laboratory research use only It is not intended for diagnostic therapeutic clinical household or any other applications IMPIOO44A2 FCCAC 00001 Page 3 of 8 cp cyagen PRODUCT APPLICATIONS OriCell Fisher 344 F344 Rat Cortical Astrocytes can be used to study neurobiology such as the maintenance of the extracellular environment in the CNS and the maintenance of neuronal metabolism and neurotransmitter synthesis as well as drug discovery in the areasof Parkinson s Huntington s and Alzheimer s Disease GENERAL HANDLING PRINCIPLES 1 A 1 Aseptic handling of the product is necessary throughout 2 For general maintenance of cells we recommend the seeding density to b
7. ove the medium and replace it with the warmed fresh medium and return the flask to the incubator 2 Avoid repeated warming and cooling of the medium If the entire contents are not needed for a single procedure transfer only the required volume to a sterile secondary container Fig 10riCell Fisher 344 Rat Cortical Astrocytes are establishe IMPIOO44A2 FCCAC 00001 Page 5 of 8 Cp cyagen PASSAGING OriCell Fisher 344 F344 RAT CORTICAL ASTROCYTES Materials Required e Trypsin EDTA Cat No TEDTA 10001 e Phosphate Buffered Saline 1xPBS Cat No PBS 10001 e OriCell Fisher 344Rat Cortical Astrocytes Cat No FCCAC 00001 e OriCell Astrocyte Growth Medium Cat No GXXAC 90011 Passaging of OriCell F344 Rat Cortical Astrocytes 1 Pre warm the OriCell Astrocyte Growth Medium 1xPBS Trypsin EDTA solution to 37 C 2 Carefully aspirate spent medium from the 80 90 confluent monolayer of OriCell F344 Rat Cortical Astrocytes 3 Add 1xPBS 6mL for T75 flask 3mLfor T25 flask Be careful not to disturb the monolayer Gently rock the flask back and forth to rinse the monolayer 4 Aspirate 1xPBS and discard Repeat the step 3 4 two or three times 6 Add Trypsin EDTA solution 1 5mL for T75 flask 0 5mL for T25 flask Gently rock theflask back and forth to ensure that the entire monolayer is covered with the Trypsin EDTAsolution Allow the trypsinization to continue until the majority of the cells approx
8. re medium whenever themediumbecomesacidic even ifthecellsdonotreach80 90 confluency In general change the growth medium every three days Time to Subculture WhenOriCell F344RatCorticalAstrocytes are 80 90 confluent itisrecommended that the cells be subcultured Do not let OriCell F344Rat Cortical Astrocytes overgrow as it will result in contact inhibition CRYOPRESERVATION OF OriCell F344 RAT CORTICAL ASTROCYTES A Note Change the culture medium with fresh growth medium 24 hours before freezing 1 Collect cells that are in logarithmic growth phase Perform a cell count to determine the viable cell density 2 Centrifuge the cells 3 5 minutes at 250xg 20 C Remove and discard the Supernatant using a pipette 3 Resuspend the cell pellet in the OriCell Cryopreservation Medium Cat No CRYO 10001 at a cell density of 10 10 cells mL 4 Dispense aliquots of the cell suspension into cryogenic storage vials that are properly labeled 5 Place the vials in a programmed controlled rate freezing apparatus and make sure the cooling rate is 1 C min 6 After 24 hours transfer the frozen vials to liquid nitrogen for long term preservation IMPIOO44A2 FCCAC 00001 Page 7 of 8 APPENDIX Related products Product Trypsin EDTA Phosphate Buffered Saline 1 x PBS OriCell F344 Rat Cortical Astrocytes OriCell Astrocyte Growth Medium References cp cyagen Catalog Number TEDTA 10001 PBS 10001 FCCAC 00001
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