Posi ve Control Cells. Part Number MA001SK

Transcription

1 Posi ve Control Cells Part Number May 18, 2015

2 Table of Contents Sec on Page About This Manual 2 Kit Contents 3 Materials Required 3 Introduc on 4 Protocol: Thawing and Ini al Pla ng 5 Example Results and Karyotype 6 Legal Informa on 7 Contact Informa on 8 About This Manual This manual assumes that the user has experience with mammalian cell culture, including the culture of primary human cells. We strongly recommend reading through the en re manual before thawing the Posi ve Control Cells. Technical support can be obtained by at techsupport@factorbio.com. Explana on of Keywords and Symbols Used in This Manual: NOTE! CAUTION! Important informa on specific to this kit. A step that may be technically challenging and/or a step that, if performed incorrectly, can cause the experiment to fail. 2

3 Kit Contents Posi ve Control Cells 10 5 human fibroblasts in 0.5mL of cryopreserva on medium NOTE! Posi ve Control Cells are shipped on dry ice. Upon receipt, immediately place cells in culture or transfer to liquid nitrogen for long term storage. Materials Required Cell culture incubator (37 C, 5% CO 2 required, 5% O 2 strongly recommended) Cell culture plas cware Cell dissocia on solu on for passaging (e.g., trypsin) Fibroblast pla ng medium (e.g., DMEM + 10% FBS) 3

4 Introduc on Posi ve Control Cells (Part Number ) are low passage human dermal fibroblasts obtained from a healthy, 32 year old male pa ent. The cells exhibit a fibroblas c morphology (see Fig. 1 on Page 6), a normal karyotype (see Fig. 2 on Page 6), and a popula on doubling me of approximately 24 hours when cultured in DMEM (high glucose) + 10% FBS at 37C in a 5%CO 2, 5%O 2 atmosphere. Posi ve Control Cells test nega ve for mycoplasma using both standard DNAfluorochrome and direct culture assays. Reprogramming the Posi ve Control Cells using the FactorStem Reprogramming Kit (sold separately) typically yields between 20 and 100 pickable colonies per well of a 6 well plate from 1000 star ng cells, corresponding to a reprogramming efficiency of 2 10% (see FactorStem Reprogramming Kit manual for details). 4

5 Protocol: Thawing and Ini al Pla ng 1. Thaw the tube of Posi ve Control Cells in a 37C waterbath, moving the tube back and forth gently to speed thawing. CAUTION! Keep the water level below the cap to avoid contamina on. 2. Once thawed, spray the outside of the tube with 70% ethanol or 70% isopropanol and place in the biosafety cabinet. 3. Using a 1000µL pipet, transfer the en re contents of the tube (approximately 500µL) to a sterile, 15mL tube. 4. Add 12mL of warm fibroblast culture medium (DMEM + 10% FBS) dropwise, with agita on. 5. Transfer the cells to a 10cm cell culture dish and place in the incubator. It is not necessary to remove the cryoprotectant. Cells will be ready to passage in 2 days. NOTE! Passage cells using standard techniques. Replace medium or passage cells every 2 days. When cultured under these condi ons, cells maintain a high popula on doubling rate for at least 2 4 passages. 5

6 Example Results and Karyotype Figure 1. Posi ve Control Cells exhibit a fibroblas c morphology when grown on uncoated cell culture plates. Figure 2. Posi ve Control Cells exhibit a normal karyotype (46,XY[20]). 6

7 Legal Informa on Limited Use License The FactorStem Reprogramming Kit, FactorStem Reprogramming RNA, FactorStem Reprogramming Medium, FactorPlex Complexa on Buffer, FactorCoat Coa ng Matrix, and Posi ve Control Cells, and their components (herea er "the products") are for research use only, and are not intended for any human or animal diagnos c or therapeu c use. By purchasing the products, the purchaser agrees to use the products to reprogram cells for in vitro research use only, and to not use the products or cells treated with or produced using the products for any other use, including any human in vivo or diagnos c use, for any screening use or for any commercial use, including any manufacturing use. The user also agrees not to subject the products to chemical or other analysis (i.e. "reverse engineering") or to sequence, amplify or a empt to sequence or amplify the products. The user agrees not to sell, transfer or otherwise make available the products or cells treated with or produced using the products (including any and all cells derived from cells treated with or produced using the products) to any third party without the express prior wri en permission of Factor Bioscience Inc. Use of the products or any part thereof for any purpose whatsoever cons tutes acceptance of these terms. If the terms of this limited use license are not accepted, the original purchaser may return the products unopened within 30 days of receipt for a full refund. Limita on of Liability No warranty, either express or implied, is provided with the products, and Factor Bioscience Inc. assumes no liability for any loss, damage or injury resul ng from their use. The products and cells treated with and/or produced using the products should be handled in accordance with all applicable safety prac ces, included those that may be required by law. Factor Bioscience Inc. makes no warranty of reprogramming speed, efficiency, effec veness or that the products will successfully reprogram the user's cells. Factor Bioscience Inc. agrees to provide the original purchaser of the FactorStem Reprogramming Kit with technical support, free of charge, for a period of one (1) year from the original date of purchase, which shall include feedback on data provided to Factor Bioscience Inc. by the original purchaser, such as images of the user's cells treated with the products. Intellectual Property The products and their use are protected by U.S. Pat. No. 8,497,124, which is owned by Factor Bioscience Inc., and by mul ple pending U.S. and foreign patents including PCT/US2012/ Purchase of the products includes a limited, non transferrable license for the original purchaser to use the products to prac ce the inven ons disclosed in these issued and pending patents for a period of one (1) year from the original date of purchase only in the manner and for the purpose described on this page under the heading "Limited Use License". Any other use of the products may infringe these and/or other issued or pending patents. Factor Bioscience Inc. assumes no liability for customer's infringement of intellectual property, including intellectual property owned by third par es. Contact Factor Bioscience Inc. at or licensing@factorbio.com for addi onal licensing informa on. FactorStem is a trademark of Factor Bioscience Inc. Lipofectamine is a registered trademark of Thermo Fisher Scien fic, Inc. 7

8 Contact Informa on Sales: Customer Support: Technical Support: Licensing: All Other Inquiries: Phone: (781) Address: Factor Bioscience Inc Cambridge Street, Suite 10B Cambridge, MA