3T3-L1 Adipocyte Care Manual - Zen
Contents
1. PREADIPOCYTE gt MATURE ADIPOCYTE nucleus Once the cells are 100 confluent incubate an additional 48 hours before initiating differentiation The cells require this time to initiate growth arrest Rev May 2010 Page 7 of 9 ZenBio Inc EXPANSION OF 3T3 L1 PREADIPOCYTES Cryopreserved 313 L1 Preadipocytes Catalog SP L1 F 10 Remove cells from liquid nitrogen and place immediately into a 37 C water bath with agitation Be careful not to submerge the cap of the vial into water Do not leave the vials in water bath after most of the content has thawed Rinse the vials with 70 ethanol before taking them to the culture hood Upon the thawing add the cells to a sterile conical bottom centrifuge tube containing 10 ml of 3T3 L1 Preadipocyte Medium PM 1 L1 Centrifuge at 280 x g 20 C 5 minutes Aspirate the medium and resuspend cells in a volume of PM 1 appropriate for counting the cells Count using a hemacytometer Place approximately 3 000 5 000 cells cm in tissue culture treated cultureware using 3T3 L1 Preadipocyte Medium PM 1 L1 Incubate cells until they are 80 85 confluent in about 5 6 days Do not let the cells become 100 confluent Cells will need to be fed every other day with PM 1 L1 Aspirate medium and wash preadipocytes 4 5 times using sterile Phosphate Buffered Saline PBS to remove all traces of serum until there is no foaming of the medium Remove the PBS and relea2. Use cells of a lower passage number The grow too many times 3T3 L1 cell line is NOT immortalized and is suitable only until passage 13 Cells will arrive at Passage 8 3T3 L1 cells grow faster in an incubator set to 10 CO Edge effects Medium in outside wells Ensure a saturated humidity in the incubator evaporated and feed the cells no less than every 3 days FREQUENTLY ASKED QUESTIONS QUESTION ANSWER What is the formulation of Zen Bio s Zen Bio s serum free media are not enhanced to supplement serum free media the absence of serum These media are available for assay procedures where cells are rested from serum Should antibiotics be included in the Yes Antibiotics and anti fungal agents are always medium recommended since the cells are primary cells All Zen Bio media contain antibiotics and anti fungal agents except Basal Medium BM 1 L1 When do the cells differentiate Lipid droplets should appear within 4 7 days after differentiation is induced They look extremely small initially Lipid accumulation continues throughout the first two weeks The lipid droplets gradually fuse to several big locules See Figures 1 amp 2 Do you provide ready to use plated No At this time they are too sensitive to the stresses of 3T3 L1 adipocytes shipping during differentiation Only cryopreserved and sub confluent plated preadipocytes are provided as plated cells What plated formats do you provide We provide 3T3 L1 preadipoc
3. zenbio com Rev May 2010 Page 1 of 9 ZenBio Inc CONTENTS PAGE Introduction 3T3 L1 Media Compositions Maintenance of Plated 3T3 L1 Preadipocytes Differentiation of 3T3 L1 Preadipocytes into Adipocytes Expansion of 3T3 L1 Preadipocytes Troubleshooting Guide oo O OORA Ww Frequently Asked Questions Rev May 2010 Page 2 of 9 ZenBio Inc INTRODUCTION 3T3 L1 adipocytes have been fundamental in metabolic disease research for 30 years Originally derived from Swiss mouse embryo tissue by Dr Howard Green of Harvard Medical School the 3T3 L1 system has been pivotal in advancing the understanding of basic cellular mechanisms associated with diabetes obesity and related disorders MATERIALS PROVIDED FOR EACH CATALOG ITEM 31T3 L1 Preadipocytes Cat SP 2096 SP 2048 SP 2024 SP 2012 e Subconfluent cells Cryopreserved 3T3 L1 preadipocytes catalog SP L1 F o Frozen vial containing at least 0 5 x10 preadipocytes store in liquid nitrogen upon receipt 50 ml 3T3 L1 Preadipocyte Medium cat PM 1 L1 Rev May 2010 Page 3 of 9 ZenBio Inc 3T3 L1 MEDIA COMPOSTIONS 3T3 L1 Adipocyte Medium 3T3 L1 Preadipocyte Medium cat AM 1 L1 cat PM 1 L1 DMEM Ham s F 12 medium 1 1 v v DMEM high glucose HEPES pH 7 4 HEPES pH 7 4 Fetal Bovine Serum FBS Bovine Calf Serum BCS Biotin Penicillin Pantothenate Streptomycin Human insulin Amphotericin B Dexamethasone Penicillin Streptomycin Amphote
4. ON INTO THE PLATE Use scissors to snip open the bag at any end The vacuum seal should be released at this time You may notice some bubbling of the medium in the plate at this time This is normal and will not affect cell performance In a sterile environment remove the plate from the bag taking care to not disturb the cover top from the plate Open the lid and remove the white liner using sterile forceps or a hemostat and discard Carefully remove the clear adhesive seal by grabbing the edge with sterile forceps or hemostat and lifting the film slowly towards the other end Discard adhesive film in appropriate biohazard waste container Replace lid on plate The excess medium added to each well for shipping should be removed before incubation in a humidified atmosphere CO incubator Depending upon the plate configuration please use the chart below to determine medium volume to remove from each well Cultureware Total shipping volume per well Removal volume per well 96 well plates 300 ul well 150 ul 48 well plates 3 miwell 24 well plates 3 0 milwell 12 well plates 5 8 milwell 5 Keep the plates at 37 C with 5 CO in a humidified incubator until ready for use The cells should be fed with 3T3 L1 Preadipocyte Medium PM 1 L1 every 2 3 days until confluent See page 6 for differentiation protocol Rev May 2010 Page 5 of 9 ZenBio Inc DIFFERENTIATION OF 3T3 L1 PREADIPOCYTES INTO ADIPOCYTES Cryopreserved 31T3 L1 Preadipocyte
5. ZenBio Inc zenbio gt 3T3 L1 Cell Care Manual Maintenance and Differentiation of 31T3 L1 Preadipocytes to Adipocytes INSTRUCTION MANUAL ZBMO0009 02 SHIPPING CONDITIONS Orders are delivered via Federal Express courier All US and Canada orders are shipped via Federal Express Priority service and are usually received the next day International orders are usually received in 3 4 days Must be processed upon shipment receipt STORAGE CONDITIONS Media Short Term 4 C 6 months 20 C All Zen Bio Inc products are for research use only Not approved for human or veterinary use or for use in diagnostic or clinical procedures LIMITED PRODUCT WARRANTY This warranty limits our liability to replacement of this product No other warranties of any kind expressed or implied including without limitation implied warranties of merchantability or fitness for a particular purpose are provided by Zen Bio Inc Zen Bio Inc shall have no liability for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product ORDERING INFORMATION AND TECHNICAL SERVICES ZenBio Inc 3200 Chapel Hill Nelson Blvd Suite 104 PO Box 13888 Research Triangle Park NC 27709 U S A Telephone 919 547 0692 Facsimile FAX 919 547 0693 Toll free continental US only 1 866 ADIPOSE _ 1 866 234 7673 Electronic mail e mail information zenbio com World Wide Web http www
6. ed cells every 2 3 days using 3T3 L1 Adipocyte Maintenance Medium until ready for assay 31T3 L1 adipocytes are suitable for most assays 7 14 days post differentiation see Figure 1 and Figure 2 Rev May 2010 Page 6 of 9 ZenBio Inc Table 1 Feeding Volumes Change PM 1 L1 to Change PM 1 L1 to Change DM 2 L1 to Change AM 1 L1 to PM 1 L1 DM 2 L1 AM 1 L1 AM 1 L1 P OUT N f ut N_ Oout N_ OUT N 96 well plate 90ul well 90ul well 150ul well 150 pl well 90 ul well 1201 well 90 ul well 120ul well 48 well plate 300 ul well 300 tl well 500u well 500 ul well 300 ul well 400 ul well 300 ul well 400 ul well Figure 1 Lipid accumulation in 3T3 L1 cells cultured in Zen Bio media 3T3 L1 preadipocytes were seeded in 24 well plates and induced to differentiate 2 days post confluent using Zen Bio s DM 2 L1 for 3 days Cells were then fed Zen Bio s AM 1 L1 with fresh media being added every other day Phase contrast images were taken on day 7 Panel A and day 14 Panel B of differentiation using an Olympus IX60 microscope equipped with a STOP digital camera 20X magnification Figure 2 3T3 L1 Growth and Differentiation Feeding Schedule DAY DAY DAY DAY DAY DAY DAY DAY DAY a2 0 3 5 TAi 9 11 13 15 proliferation gt lt 48 hrs Feed Feed Feed 100 Feed Feed Feed Feed Feed Feed Feed PM 1 L1 PM 1 L1 PM 1 L1 confluent DM 2 L1 AM 1 L1 AM 1 L1 AM 1 L1 AM 1 L1 AM 1 L1 AM 1 L1
7. ricin B 3T3 L1 Differentiation Medium 3T3 L1 Basal Medium cat DM 2 L1 cat BM 1 L1 DMEM Ham s F 12 medium 1 1 v v DMEM Ham s F 12 medium 1 1 v v HEPES pH 7 4 HEPES pH 7 4 Fetal Bovine Serum FBS Biotin Biotin Pantothenate Pantothenate Human insulin Dexamethasone Penicillin Streptomycin Amphotericin B Isobutylmethylxanthine PPARy agonist NOTE All media except cat PM 1 L1 contain 3 15g L D glucose PM 1 L1 contains 4 5g L D glucose All media are also available without serum and or phenol red free Please inquire for custom media requests MEDIA EXPIRATION DATES e If placed at 4 C upon arrival the media is stable until the expiration date on the bottle label e If stored at 20 C upon arrival it is stable for 6 months Add fresh antibiotics when you are ready to use Rev May 2010 Page 4 of 9 ZenBio Inc MAINTENANCE OF PLATED 3T3 L1 PREADIPOCYTES Your 3T3 L1 preadipocytes have arrived in our patented CellPorter packaging system Upon receiving the plates please follow the instructions carefully to ensure your safety and the optimal performance of these cells 1 Check the seal for each plate Discard any plate where the vacuum seal has been compromised during shipment ALWAYS WEAR GLOVES AND USE PROTECTIVE MEASURES WHEN HANDLING CULTURED CELLS Place the package into a sterile environment THIS IS VERY IMPORTANT SINCE BREAKING THE VACUUM SEAL MAY POTENTIALLY INTRODUCE CONTAMINATI
8. s Catalog SP L1 F 1 Remove cells from liquid nitrogen and place immediately into a 37 C water bath with agitation Be careful not to submerge the cap of the vial into water Do not leave the vials in water bath after most of the content has thawed Rinse the vials with 70 ethanol before taking them to the culture hood 2 Upon the thawing add the cells to a sterile conical bottom centrifuge tube containing 10 ml of 3T3 L1 Preadipocyte Medium PM 1 L1 3 Centrifuge at 280 x g 20 C 5 minutes Aspirate the medium and resuspend cells in a volume of PM 1 L1 appropriate for counting the cells Count using a hemacytometer 4 Place approximately 5 000 cells cm in tissue culture treated cultureware using 3T3 L1 Preadipocyte Medium PM 1 L1 5 Maintain cells until they are 100 confluent in about 6 7 days in a humidified incubator 37 C with 5 10 COs Cells will need to be fed every other day with PM 1 L1 during this time See Table 1 for feeding volumes 6 Once the cells are confluent incubate an additional 48 hours before initiating differentiation 7 Two days after the cells have been confluent remove the 3T3 L1 Preadipocyte Medium cat PM 1 L1 and replace with an appropriate volume 3T3 L1 Differentiation Medium cat DM 2 L1 see table 1 below for recommended volumes Incubate for 3 days 8 Remove the 31T3 L1 Differentiation Medium and replace with 3T3 L1 Adipocyte Maintenance Medium Incubate for 2 3 days 9 Fe
9. se the cells from the bottom of the cultureware vessel by adding 30ul cm of 0 25 trypsin 2 21mM EDTA solution cat TRP 100 Allow cells to trypsinize for 5 minutes at 37 C Tap the flask gently to loosen the cells Neutralize the trypsin using at least 100pl cm 3T3 L1 Preadipocyte Medium cat PM 1 L1 Check the vessel under a microscope to ensure all cells are free of the flask bottom Count the cells and plate in desired format see page 6 Ensure cells are evenly suspended when plating large numbers of plates or flasks Place in a humidified incubator at 37 C and 5 10 COz making sure the surface is level for even cell distribution Follow the differentiation protocol as outlined on pages 6 7 We DO NOT recommend expanding the preadipocytes that are older than Passage 13 Cells will arrive at Passage 8 Rev May 2010 Page 8 of 9 ZenBio Inc TROUBLESHOOTING GUIDE Observation Possible causes Suggestions Preadipocytes do not Cells have been passaged Use cells of a lower passage number The differentiate well too many times 3T3 L1 cell line is NOT immortalized and is suitable for only 12 13 passages Ensure cells are 100 confluent for 48 hours prior to initiating differentiation Do not use fetal bovine serum during the proliferation process It will affect later differentiation potential We recommend using Zen Bio s 3T3 L1 Preadipocyte Medium cat PM 1 L1 Preadipocytes do not Cells have been passaged
10. ytes in the following formats for 3T3 L1 cells 96 well 48 well 24 well 8 chamber slides Rev May 2010 Page 9 of 9
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