User Manual - Cyagen Biosciences
Contents
1. OriCell Embryoid Body EB Formation Medium Cat No MUXES 90051 The formation of embryoid body EB is the principal step in the differentiation of ES cells When maintained in the EB formation medium and in the absence of MEF feeder layers ES cells differentiate spontaneously and then form three dimensional aggregates This structure facilitates multicellular interactions in which cell cell contact exists and gap juncitons may be established IMPI0070A3 MUBES 01201 Page 9 of 14 6 cyagen Protocol 1 10 11 12 Dissociate OriCell Strain C57BL 6 Mouse ESCs RFP by incubating the cells with trypsin solution at 379C for 1 2 min Add an appropriate volume of Cyagen OriCell EB Formation Medium e g 3 mL for each well of six well plate to stop reaction and gently pipette up and down until cells in colonies become single cells Transfer cell suspension into a 15 ml conical tube and centrifuge at 250 X g for 5 minutes to pellet the cells Carefully aspirate as much of the supernatant as possible Add appropriate amount of Cyagen OriCell EB Formation Medium to the conical tube and gently resuspend the cells Plate cell suspension in 100 mm adherent dishes Incubate the adherent dishes in a 379C incubator for 30 40 minutes to separate Mouse Embryonic Fibroblasts from OriCell Strain C57BL 6 Mouse ESCs RFP Carefully collect the suspending OriCell Strain C57BL 6 Mouse ESCs RFP and adjust the cell concentrat2. Trypsin EDTA solution to 3 9C 2 Aspirate the spent medium from the OriCel MEF Rinse MEF with 1xPBS 3 mL for one well of six well plate Aspirate the 1xPBS from the flask and discard I ICR Mouse Embryonic Fibroblasts Repeat step 3 4 once or twice Add the pre warmed OriCell Mouse ESC Growth Medium Return the MEF to the 5 COz humidified incubator Gr Note Be careful not to disturb the monolayer of MEF during step 2 6 Carefully aspirate off spent medium from OriCell Strain C57BL 6 Mouse ESCs RFP Rinse the cells with 1xPBS 3 mL for one well of six well plate Aspirate the 1xPBS from the flask and discard 10 Repeat the step 8 9 two or three times 11 Add Trypsin EDTA solution 200 uL for one well of six well plate and incubate for 1 2 minutes until the OriCell Strain C57BL 6 Mouse ESCs RFP are dissociated At this point gently tap the side of the flask to release the majority of cells from the culture surface 12 Add OriCell Mouse ESC Growth Medium 3 mL for one well of six well plate and gently pipette up and down until colonies become dissociated to single cells IMPI0070A3 MUBES 01201 Page 8 of 14 oC cyagen Note Be careful not to introduce any bubbles 13 Transfer the dissociated cells into a 15 mL conical tube 14 Centrifuge the tube at 250 x g for 5 minutes to pellet the cells 15 Carefully aspirate off as much of the supernatant as possible 16 Add 2 mL of OriCell
3. Mouse ESC Growth Medium to the conical tube and re suspend the cells thoroughly but gently 17 Plate the cells into flasks containing the MEF Split ratios for OriCell Strain C57BL 6 Mouse ESCs RFP can vary from 1 6 to 1 10 Do not exceed 1 10 18 Add sufficient medium 19 Incubate the cells at 37 C in a 5 CO humidified incubator until it is time to split again We typically split OriCellTM Strain C57BL 6 Mouse ESCs RFP every other day Note 1 OriCell Strain C57BL 6 Mouse ESCs RFP should be plated at a density that provides an even distribution of colonies over the surface but does not result in contact between the colonies Differentiation can occur if the colonies are plated too densely or too sparsely 2 OriCell Strain C57BL 6 Mouse ESCs RFP should not be over subcultured minimize the number of passages and the length of time the cells are kept in culture This will ensure enhanced and reproducible experimental results A Hints Time to Split Strain OriCell C57BL 6 Mouse Embryonic Stem Cells With RFP Passage the cells before the colonies become too large and dense When plated at the optimum density OriCell Strain C57BL 6 Mouse ESCs RFP should be passaged every 48 hours DIFFERENTIATION OF OriCell STRAIN C57BL 6 Mouse EMBRYONIC STEM CELLS WITH RFP ESCs RFP OriCell Strain C57BL 6 Mouse ESCs RFP are capable of forming embryoid bodies in vitro and teratomas in nude mice Materials Required
4. aspirate as much of the supernatant as possible and add 3 mL of fresh OriCell Mouse ESC Growth Medium pre warmed to 37 C 11 Gently re suspend the cells in OriCell Mouse ESC Growth Medium 12 Plate the cells into TWO T25 flasks and add sufficient OriCell Strain C57BL 6 Mouse ESCs RFP Gently rock the culture flask to evenly distribute the cells 13 Incubate at 37 C in a 5 CO humidified incubator 14 The next day change the medium with fresh OriCell Strain C57BL 6 Mouse ESCs RFP pre warmed to 37 C A Note Changing Medium 1 Warm an appropriate amount of OriCell Mouse ESC Growth Medium to 37 C in a sterile container Remove the spent 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 IMPI0070A3 MUBES 01201 Page 7 of 14 cyagen Fig 4 Image of OriCell Stain C57BL 6 Mouse Embryonic Stem Cells With RFP at P21 cultured on OriCell ICR Mouse Embryonic Fibroblasts Irradiated feeder cells PASSAGING OriCell STRAIN C57BL 6 Mouse EMBRYONIC STEM CELLS WITH RFP ESCs RFP Materials Required e OriCell M Mouse Embryonic Stem Cell Growth Medium Cat No MUXES 90011 e Vessels plated with MEF Passaging C57BL 6 Mouse ESCs RFP 1 Pre warm OriCell Mouse ESC Growth Medium 1xPBS
5. Ydw LA Technical SUpport iei Wil MA GWM YW MON AAT A MAA Y WN MYD wi A 6 cyagen CONTENTS AND STORAGE PraducH Name Strain C57BL 6 Mouse Embryonic Stem Cells With RFP Catalog No MUBES 01201 Amount per Vial 1x10 Cells Cryopreserved At The 23rd 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 Embryonic Stem Cells With RFP ESCs RFP are pluripotent cells derived from the inner cell mass of blastocysts These cells are able to differentiate into all derivatives of the primary germ layers including ectoderm endoderm and mesoderm thus generating every cell type in the body Different from most other stem cells ESCs RFP are capable of self renewal indefinitely Because of their plasticity and potentially unlimited capacity for self renewal ES cell therapies have been proposed for regenerative medicine and tissue replacement OriCell Strain C57BL 6 Mouse ESCs RFP maintain diploid karyotype after extended passages in vitro These cells express specific clusters of different proteins for ESCs RFP and are capable of forming embryoid bodies in vitro and developing teratomas in nude mice Cyagen OriCell Strain C57BL 6 Mouse ESCs RFP are derived from the inner cell mass of strain C57BL 6 Mouse blastocysts at 3 5 dpc and cultured on y ray ir
6. andling of the product is necessary throughout 2 Once the cells have been established always freeze several vials of OriCell C57BL 6 Mouse ESCs RFP as a backup 3 Establish and maintain Cyagen OriCell Strain C57BL 6 Mouse Embryonic Stem Cells With RFP on mouse embryonic fibroblasts MEFs feeder layers We recommend using Cyagen OriCell Strain ICR MEFs Irradiated for culturing mouse ESCs RFP 4 For general maintenance of cells we recommend the seeding density to be 2 0 2 5 x 10 cells cm 5 Do not let OriCell C57BL 6 Mouse ESCs RFP overgrow as it will result in contact between the colonies We recommend that the Mouse ESCs RFP are routinely passaged every 48 hrs GELATIN COATING OF TISSUE CULTURE VESSELS FOR MOUSE EMBRYONIC FIBROBLASTS MEFS Materials Reguired e Gelatin Solution Cat No GLT 11301 Gelatin Coating of Tissue Culture Vessels 1 Add sufficient Gelatin Solution into the culture vessel to completely cover its base 2 Swirl until Gelatin Solution coats the entire base of vessel Let it sit for at least 30 IMPI0070A3 MUBES 01201 Page 4 of 14 A 6 cyagen 3 Aspirate off all of the Gelatin Solution and allow the residual amount to evaporate by leaving the vessel sitting open in the laminar flow hood biological safety cabinet for no more than 30 minutes minutes at room temperature 4 Enclose the culture vessel once it has dried Note Gelatinized dishes or flasks can be stored at 4 C for
7. eee 9 C y d e We help you discover hfe OriCell Strain C57BL 6 Mouse Embryonic Stem Cells With RFP ESCs RFP Cat No MUBES 01201 6 cyagen Table of Contents Contents and Storage wessecceccceecccceeeeeeeeeeeeeeeeeeeeeuseeueeauauevaueueeeuaeeuauaeaeaveeeeauavaeaeaeavenaeanas 3 Product Introduction cccccsccececeeeeeeeueeseeeueueueueaeeueueueuseuaueneuseeaueueuseeaeeueueueaeeeeueeeerennges 3 Cell Characteristics and Identity 9 99 9YYL AW LARA nesinnes aiaiai aii aaae 4 Product Applications 9YL LY ALA HLR LN RR RRRRE REF ENE RE EER a aaa END NNDR Dun uu amp General Handling Principles uu YL YLH LY GRY ainni RRR RR RL LRL RR RR REN RR RR RR Dn Ynn Enau amp Gelatin Coating of Tissue Culture Vessels for MEFS 99 99 9YY Y LY LY RR nh 4 Culturing OriCell Strain C57BL 6 Mouse ESCs RFP Thawing and Establishing of MEF Feeder Cells eu nun nun Ynn YY RR LY ER RE Hu 5 Thawing and Establishing of OriCell Strain C57BL 6 Mouse ESCs RFP 6 Passaging OriCell Strain C57BL 6 Mouse ESCs RFP e us WY Wyn 8 Differentiation of OriCell Strain C57BL 6 Mouse ESCs RFP L ueu 9 Cryopreservation of OriCell Strain C57BL 6 Mouse ESCs RFP 11 ADDED chitin a Eu HEH HETH FEE L2 Related products uii Yni GWY NN TWN YW RUN RUN Wu NWN DRA AWN yn NAU aaa AW WaR ui Ru ents LS Refenrenc6 S cei iu dU YN a UYD
8. elated products Product Catalog Number Gelatin Solution GLT 11301 OriCell Mouse Embryonic Fibroblasts MUIEF 01002 OriCell Mouse Embryonic Fibroblast Growth Medium MUXEF 90011 OriCell Strain CS7BL 6 Mouse Embryonic Stem Cells MUAES 01201 With RFP OriCell Mouse Embryonic Stem Cell Growth Medium MUXES 90011 Phosphate Buffered Saline 1xPBS PBS 10001 Trypsin EDTA TEDTA 10001 OriCellTM Embryoid Body EB Formation Medium MUXES 90051 OriCell NCR Protein Free Cryopreservation Medium NCPF 10001 IMPI0070A3 MUBES 01201 Page 13 of 14 ccyagen References G R Martin 1981 Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells PNAS 78 7634 7638 T M Magin J McWhir and D W Melton 1992 A new mouse embryonic stem cell line with good germ line contribution and gene targeting freguency Nucleic Acids Research 20 14 3795 3796 J A Thomson J Kalishman and T G Golos 1995 Isolation of a primate embryonic stem cell line PNAS 92 7844 7848 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 IMPI0070A3 MUBES 01201 Page 14 of 14
9. ion to 5 x 10 cells mL with OriCell EB Formation Medium Plate 10mL cell suspension in one 100 mm non adherent petri dish Incubate the cells at 37 C in a 5 CO humidified incubator for 5 days to form EB and change the medium every other day Plate EB into adherent surface of gelatin coated tissue culture vessels in Cyagen OriCell EB Formation Medium Incubate the EB at 37 C in a 5 CO humidified incubator for about 14 days Change media every other day Stain the differentiated cells with antibodies against endodermal mesodermal and ectodermal markers at day 14 after EB differentiation IMPI0070A3 MUBES 01201 Page 10 of 14 6 cyagel CRYOPRESERVATION OF OriCell STRAIN C57BL 6 Mouse EMBRYONIC STEM CELLS WITH RFP ESCs RFP USING OriCell NCR PROTEIN FREE CRYOPRESERVATION MEDIA OriCell NCR Protein Free Cryopreservation Medium Cat No NCPF 10001 is a protein free ready to use freezing medium Its chemically defined and protein free formulation has been optimized to stem cells and primary cells thus greatly enhancing the viability and integrity of these cells by protecting them from damage during the one step freeze thaw procedure Unlike other conventional freezing media which reguire a slow programmed freeze this product allows the cells to be directly frozen at 80 C Cryopreservation A Note Change the culture medium with fresh growth medium 24 hours before freezing 1 Collect cells that are in the l
10. medium twice and transfer all of the cells to the dish Care should be taken to avoid introducing bubbles during pipetting Also avoid vortexing and high speed centrifugation Warm medium to 37 C before recovery Discard the cells in question and disinfect the laboratory environment before recovering the next batch of cells Wash the cells with PBS 2 3 times to remove serum prior to trypsinization serum will inhibit the function of trypsin Control the digestion time Increase the plating density MEFs should be used up in 5 7days after recovery Use Cyagen tailor made culture media If other serum and media products are used please perform validation to ensure compatibility Change the medium the next day after recovery to ensure removal of all dead cells Discard the cells in question and disinfect the experimental environment before recovering Page 12 of 14 6 cyagen Some stem cells can secrete factors to support cell growth Therefore a certain degree of plating density must be maintained otherwise it will lead to cell proliferation slow down and finally cell aging _ Control the digestion time Plating density is too low DMSO is not completely Wash the cells with pre warmed medium removed during cell recovery 2 3 times during recovery ESCs RFP clone is too large Lower plating density Differentiation reagents need Use Cyagen tailor made differentiation to be optimized medium R
11. no more than 2 weeks provided they remain sterile THAWING AND ESTABLISHING MOUSE EMBRYONIC FIBROBLASTS MEFs Materials Reguired e Gelatin Solution Cat No GLT 11301 e OriCell Strain ICR Mouse Embryonic Fibroblasts Cat No MUIEF 01002 e OriCell M Mouse Embryonic Fibroblast Growth Medium Cat No MUXEF 90011 Thawing and Establishing MEFs Pre warm the OriCell MEF Growth Medium to 37 C 2 Add 9 mL of OriCell MEF Growth Medium to a 15 mL conical tube Remove the cryovial of OriCell Strain ICR MEFs from liquid nitrogen Quickly thaw the vial in a 37 C water bath until the last ice crystal disappears For optimal results be sure to 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 4 As soon as the cells are completely thawed disinfect the outside of the vial with 70 v v ethanol 5 Use a pipette to transfer the cells to the 15 mL conical tube containing OriCell MEF Growth Medium inside a biosafety cabinet Be careful not to introduce any bubbles during the transfer process 6 Rinse the vial with 1 mL of medium to reduce cell loss Subsequently transfer this 1mL of cell suspension to the conical tube 7 Gently mix the cell suspension by slowly pipetting up and down Be careful not to introd
12. ogarithmic growth phase Perform a cell count to determine the viable cell density 2 Centrifuge the cells for 3 5 minutes at 250 x g and 20 C Remove and discard the supernatant using a pipette 3 Resuspend the cell pellet in the OriCell NCR Protein Free Cryopreservation Medium 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 directly in a 80 C freezer After 24 hours transfer the frozen vials to liquid nitrogen for long term preservation IMPI0070A3 MUBES 01201 Page 11 of 14 Cyagen The table below lists some potential problems and solutions for culturing OriCell Strain C57BL 6 Mouse Embryonic Stem Cells With RFP Problem Low cell recovery rate Slow cell growth Cell aging IMPI0070A3 MUBES 01201 Cause The storage condition does not meet the requirements Thawing the cells takes too long time Cells are incompletely recovered after thawing Cells are handled roughly Medium is not pre warmed Mycoplasma contamination Over digestion Plating density is too low MEFs have been cultured for too long Inappropriate serum and medium Dead cells are not removed promptly Cell Contamination Solution Purchase a replacement and store in liquid nitrogen for long term preservation Thaw cells for no more than 3 minutes After aspirating off medium wash the tube with culture
13. onic Stem Cells With RFP Cat No MUAES 01001 e OriCell M Mouse Embryonic Stem Cell Growth Medium Cat No MUXES 90011 Thawing and Establishing C57BL 6 Mouse ESCs RFP IMPI0070A3 MUBES 01201 Page 6 of 14 6 cyagen 1 Pre warm the OriCell Mouse ESC Growth Medium and 1xPBS to 37 C 2 Add 9 mL of OriCell Mouse ESC Growth Medium to a 15 mL conical tube Remove the cryovial of OriCell Strain C57BL 6 Mouse ESCs RFP from liquid nitrogen 4 Quickly thaw the vial in 37 C water bath until the last ice piece disappears For optimal results be sure to 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 A Note Results will be less than optimal if the cells are thawed for more than 3 minutes 5 As soon as the cells are completely thawed disinfect the outside of the vial with 70 v v ethanol 6 In a laminar flow hood use pipette to transfer the cells to the conical tube containing OriCell Mouse ESC Growth Medium A Note Be careful not to introduce any bubbles during the transfer process 7 Rinse the vial with 1 ml of medium to reduce the loss of cell and then transfer the 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 suspensions at 250 x g for 5 minutes 10 Carefully
14. radiated mouse embryonic fibroblasts as feeder cells in OriCell Mouse ESC Growth Medium and then have been transfected with a lentiviral construct containing a RFP expression motif and been selected from a purified ESCs RFP clone In addition these cells have been tested for e Exogenous Factors bacterial fungal contamination mycoplasma contamination and endotoxin contamination e Characteristics post thaw viability cell cycle verification of undifferentiated state and differentiation potential This product is intended for laboratory research use only It is not intended for diagnostic therapeutic clinical household or any other applications IMPI0070A3 MUBES 01201 Page 3 of 14 6 cyagel CELL CHARACTERISTICS AND IDENTITY e Ability to differentiate into all derivatives of the three primary germ layers e Reproduce indefinitely under proper conditions e Positive for pluripotent stem cell markers Oct4 SSEA 1 and Nanog 90 negative for SSEA 3 and SSEA 4 lt 5 PRODUCT APPLICATIONS OriCell Strain C57BL 6 Mouse Embryonic Stem Cells With RFP ESCs RFP are potent tools for basic and applied research in diverse fields including basic mechanism involved in developmental procedure and disorder regenerative biology and potential therapies Specially ESCs RFP are a valuable utility to make genetically modified mice by introducing mutations into the mouse germ line GENERAL HANDLING PRINCIPLES 1 Aseptic h
15. uce any bubbles Centrifuge the cell suspension at 250 x g for 5 minutes Carefully aspirate off as much of the supernatant as possible and add 3 mL of fresh OriCell MEF Growth Medium pre warmed to 37 C 10 Gently resuspend the cells in OriCell MEF Growth Medium 11 Seed the cells into 6 well plates pre coated with Gelatin Solution or other IMPI0070A3 MUBES 01201 Page 5 of 14 6 cyagen appropriate flasks and add sufficient OriCell MEF Growth Medium Gently rock the culture plate to evenly distribute the cells A Note We recommend the seeding density of MEFs to be 2 0 3 0x10 cells cm7 12 Incubate at 37 C in a 5 CO humidified incubator 13 The next day change the medium with fresh OriCell MEF Growth Medium pre warmed to 379C A Note 1 If the next day thawing of the Embryonic Stem Cells With RFP is performed the medium can be changed directly to embryonic stem cell growth medium 2 Thawing the feeder cells should be performed at least one day before thawing Embryonic Stem Cells With RFP 3 The feeder cells should be used as soon as possible once thawed y K WA 1 Fig 1 Cyagen OriCell Mouse Embryonic Fibroblasts Irradiated plated on culture vessels coated with 0 1 gelatin THAWING AND ESTABLISHING OriCell STRAIN C57BL 6 Mouse EMBRYONIC STEM CELLS WITH RFP ESCs RFP Materials Required e Gelatin Solution Cat No GLT 11301 e OriCell M Strain C57BL 6 Mouse Embry
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