PH10205X HEK Cells Manual
Contents
1. PrimaPure Human Epidermal Keratinocytes HEK Catalog Description Content Amount PH10205A HEK Adult gt 500 000 cells Related Products Keratinocyte Serum Free Growth Medium 500 ml US Genlantis A division of Gene Therapy Systems Inc PM131500 PH10205F HEK Fetal gt 500 000 cells Keratinocyte Calcium Free Growth Medium 500 ml PM133500 PH10205N HEK Neonatal gt 500 000 cells HEPES Buffered Saline Solution HBSS 100 ml PR062100 PH10205AK HEK Adult Complete System 1 Kit Trpsin EDTA 100 ml PRO70100 PH10205FK HEK Fetal Complete System 1 Kit Trypsin Neutralizing Solution 100 ml PR080100 PH10205NK HEK Neonatal Complete System 1 Kit Each kit contains an ampoule of cryopreserved HEK PH10205A PH10205F or PH10205N 500 ml of Keratinocyte Serum Free Growth Medium PM131500 and a Subculture Reagent Kit PRO90100K INTRODUCTION Human Epidermal Keratinocytes HEK are primary cells isolated from normal human neonatal foreskin or adult skin They are cryopreserved at the end of primary culture and can be cultured and propagated at least 16 population doublings HEK is a suitable in vitro model for dermal toxicology tests in screening new skin products and for developing dermal skin substitutes HEK has been used in numerous applications including investigations of human epidermal development differentiation and cellular aspects of2. ar research use only excludes and without limitation resale repackaging or use for the making or selling of any commercial product or service without the written approval of Genlantis Separate licenses are available for non research use or applications The PrimaPure cells and reagents are not to be used for human diagnostic or included used in any drug intended for human use Care and attention should be exercised in handling the product by following appropriate research lab practices Purchasers may refuse this license by returning the enclosed materials unused By keeping or using the enclosed materials you agree to be bound by the terms of this license The laws of the State of California shall govern the interpretation and enforcement of the terms of this license JL060514 Genlantis Page 2 of 2 10190 Telesis Court San Diego CA 92121 Toll Free 888 428 0558 e U S amp Canada 858 457 1919 e www genlantis com 2006 Gelantis and Gene Therapy Sytems Inc
3. bculturing HEK Cells 11 Aspirate the Trypsin EDTA solution 1 Remove above solutions from the 20 C freezer and thaw 12 Re cap the flask and place in a 37 C incubator for 2 minutes overnight in a refrigerator 13 Release the rounded cells from the culture surface by hitting 2 Make sure all the reagents are thawed Swirl each bottle the side of the flask against your palm until most of the cells gently several times to form homogeneous solutions are detached 3 Store all reagents at 4 C for future use The activity of 14 If the cells are not visually detached place the flask into the Trypsin EDTA Solution will be stable for 2 weeks when stored incubator for an additional thirty seconds then repeat step 13 at 4 C 15 Pipette 5 ml of Trypsin Neutralizing Solution to the flask to 4 Aliquot Trypsin EDTA solution and store the unused portion at inhibit further tryptic activity 20 C if only portion of the Trypsin EDTA is needed 16 Transfer the cell suspension from the flask to a 50 ml sterile 5 Take the Keratinocyte Growth Medium Cat No PM131500 or conical tube p l 2 Cat No PM133500 from the refrigerator Decontaminate the 17 Rinse the flask with an additional 5 ml of Trypsin Neutralizing bottle with 70 alcohol in a sterile hood Solution and transfer the solution into the same conical tube 6 Pipette 35ml of Keratinocyte Growth Medium to a T 175 flask 18 Examine the T 75 flask under a microscope If there are gt 20 8 Wash th
4. e monolayer of cells with HBSS and remove the the cells nee solution by aspiration 20 Aspirate the supernatant from the tube without disturbing the 9 Pipette 8 ml of Trypsin EDTA Solution into the T 75 flask cell pellet l l l Rock the flask gently to ensure the solution covers all the 21 Flick the tip of the conical tube with your finger to loosen the cells cell pellet NOTE Trypsinize Cells at Room Temperature Do Not Warm 22 Resuspend the cells in 2 ml of Keratinocyte Growth Medium Any Reagents to 37 C by gently pipetting the cells to break up the clumps Transfer y Reag l 10 Re cap the flask tightly and monitor the trypsinization progress 23 rate ges ee cs ai t l t at room temperature under an inverted microscope and over a o ee alae ee eee 60 seconds period Inoculate at 7 500 cells per cm for rapid growth or at 5 000 cells per cm for regular subculturing REFERENCES 1 Delco V et al Alternative Methods in Toxicology 3 466 1985 2 Joyce S T et al J Biomed Mater Res 22 937 1988 3 Holbrook K A Biochemistry and Physiology of the Skin Vol 1 64 1983 4 Fuchs F J Cell Biol 111 No 6 Pt 2 2807 1990 5 Koizami H et al British J Dermatol 134 686 1996 LICENSE The purchase price paid for the PrimaPure cells and reagents grants end users a non transferable non exclusive license to use the kit and or its components for internal research use only as described in this manual in particul
5. nt 0 75 ml 7202007 GeneSilencer siRNA Transfection Reagent 200 reactions 7500750 Turn the vial cap a quarter turn to release any liquid nitrogen that may be trapped in the threads then re tighten the cap Thaw the cells quickly by placing the lower half of the vial in a 37 C water bath for 1 2 minutes Take the vial out of the water bath and wipe dry Decontaminate the vial exterior with 70 alcohol in a sterile Biological Safety Cabinet Remove the vial cap carefully Do not touch the rim of the cap or the vial Resuspend the cells in the vial by gently pipetting the cells 5 times with a 2 ml pipette Be careful not to pipette too vigorously as to cause foaming Pipette the cell suspension 1ml from the vial into the T 75 flask containing 15 ml of Keratinocyte Growth Medium Cap the flask and rock gently to evenly distribute the cells Place the T 75 flask in a 37 C 5 CO2 humidified incubator Loosen the cap to allow gas exchange For best results do not disturb the culture for 24 hours after inoculation Change to fresh Keratinocyte Growth Medium after 24 hours or overnight to remove all traces of DMSO Change Keratinocyte Growth Medium every other day until the cells reach 45 confluent Double the Keratinocyte Growth Medium volume when the culture is gt 45 confluent or for weekend feedings Subculture the cells when the HEK reach 60 80 confluent Human Epidermal Keratinocytes HEK Manual Ill Su
6. skin diseases MATERIALS AND METHODS Preparation for Culturing 1 2 Make sure your Class II Biological Safety Cabinet with HEPA filtered laminar airflow is in proper working condition Clean the Biological Safety Cabinet with 70 alcohol to ensure it is sterile Turn the Biological Safety Cabinet blower on for 10 min before cell culture work Make sure all serological pipettes pipette tips and reagent solutions are sterile Follow the standard sterilization technique and safety rules a Do not pipette with mouth b Always wear gloves and safety glasses when working with human cells even though all the strains have been tested negative for HIV Hepatitis B and Hepatitis C c Handle all cell culture work in a sterile hood Il Culturing HEK Cells 1 Take the Keratinocyte Growth Medium Catalog PM131500 or PM133500 from the refrigerator Decontaminate the bottle with 70 alcohol in a sterile hood Pipette 15 ml of Keratinocyte Growth Medium to a T 75 flask NOTE Keep medium to surface area ratio at 1ml per 5 cm2 Example 5 ml for a T 25 flask or a 60 mm tissue culture dish 15 ml for a T 75 flask or a 100 mm tissue culture dish Remove the cryopreserved vial of HEK from the liquid nitrogen storage tank Use proper protection for your eyes and hands Subculture Reagent Kit including100 ml each of HBSS Trpsin EDTA and Trpsin Neutralizing Solution PR090100K GenePORTER 2 Transfection Reage
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