Maintenance of Novus Cerebral Cortical Neurons

Transcription

1 Maintenance of Novus Cerebral Cortical Neurons Catalog nos. NBP , NBP , NBP thru NBP , NBP , NBP thru NBP , NBP , NBP thru NBP , NBP thru NBP Instruction Manual Version /03/2014

2 Table of Contents Contents and Storage Additional Material Required for culturing Novus hccns Preparing Matrix for adherent cell culture (NBP , NBP thru NBP ) Thawing Novus hccns (NBP , NBP thru NBP ) Maintaining Novus hccns Technical Support i

3 NOVUS CELL REAGENTS Specially formulated cell supplements to complement our neural progenitor cells and cortical neurons NBP Neural Maintenance Media Provided as 500ml/kit (4 bottles of 125mL) NBP Neural Coating Solution (orange color cap) Provided as 50X stock solution (3 x 120uL) NBP Neural Advance (red color cap) Provided as 1000X stock solution (4 x 26uL) NBP Sure Mix (purple color cap) Provided as 1000X stock solution (3 x 26uL) NBP Neural Unlock (white color cap) Provided as 1000X stock solution (1 x 25uL) NBP Sure Boost (yellow color cap) Provided as 1000X stock solution (1 x 10uL) NBP Neural Enhance (green color cap) Provided as 1000X stock solution (1 x 15uL) NBP Neural Sure Growth (blue color cap) Provided as 1000X stock solution (1 x 15uL) info@novusbio.com P:

4 Content and Storage Configurations Catalog nos. NBP , NBP , NBP thru NBP include Novus hccns (Pre-plated in 96-well plate) Catalog nos. NBP , NBP thru NBP include Novus hccns (Frozen in cryovials) Novus CellSolutionsTM Reagents Catalog no. NBP includes Neural Maintenance Medium Catalog no. NBP includes Neural Coating Solution Catalog no. NBP includes Sure MixTM Catalog no. NBP includes Sure BoostTM Catalog no. NBP includes Neural EnhanceTM Shipping Novus frozen hccns and CellSolutionsTM Reagents are shipped on dry ice. Plated hccns are shipped at room temperature in a sealed 96-well plate. 1

5 Storage Catalog nos. NBP , NBP , NBP thru NBP Open immediately and store at 37 C in 5% CO2. Catalog nos. NBP , NBP thru NBP Remove cryovials from the dry ice packaging and immediately place into liquid nitrogen storage. Alternatively, thaw and use the cells immediately. Catalog nos. NBP , NBP thru NBP , NBP thru NBP CellSolutions TM Reagents store at - 80 o C Additional Material Required for culturing Novus hccns Reagents and Plastics Water, distilled Trypan Blue Stain D-PBS Conical tubes (15 ml) 35-mm tissue culture dishes 6-well tissue culture plates Life Technologies, Cat. no Life Technologies, Cat. no Life Technologies, Cat. no BD, Cat. no Fisher Nunc, Cat. no Fisher Nunc, Cat. no Pipette tips Serological pipettes (5 ml, 10 ml) Medium Reservoirs (for NBP , NBP thru NBP , NBP thru NBP ) Starlab, Cat. no. S BD, Cat. no , Vistalab Technologies Cat. no D 2

6 Equipment Vertical laminar flow hood certified for Level II handling of biological materials (Class II Biosafety Cabinet) Incubator with humidity and gas control to maintain 37 C and >90% humidity in an atmosphere of 5% CO 2 in air Tissue culture centrifuge (e.g. Eppendorf 5810) Pipette-aid Micropipettes Hemacytometer Inverted microscope with 4X, 10X and 20X phase objectives (e.g. Olympus CKX31) 37 C water bath Multichannel pipettes (for culturing NBP , NBP , NBP thru NBP ) 3

7 Preparing Matrix for Adherent Cell Culture (NBP , NBP thru NBP ) 1. Thaw the Neural (NBP ) Coating solution at 4 C. 2. Dilute the Neural Coating stock solution (50X) in D-PBS (without calcium or magnesium) to make working solution. 3. Coat the surface of your cell culture vessel with the Neural Coating working solution at a concentration of 100μL per cm Incubate the culture vessel overnight at 37 C. 4

8 Thawing Novus hccns (NBP , NBP thru NBP ) 1. Remove the cells from dry ice or liquid nitrogen storage, and immediately transfer the cells to a 37 C water bath. 2. Quickly thaw the vial of cells by swirling it in the 37 C water bath. Remove the vial before the last bit of ice has melted. Do not submerge the vial completely. 3. When thawed, immediately transfer the cells into a 15-ml sterile conical tube, and carefully add 10 ml of pre-warmed Neural Maintenance Medium. 4. Centrifuge the cells at 200 g for 5 mins, and discard the supernatant. 5. Resuspend the cell pellet in Neural Maintenance Medium supplemented with 1000X Sure Boost TM (NBP ) stock solution (i.e. 1µl Sure Boost TM per ml medium). 6. Plate the resuspended cells at no less than 50,000 cells/cm 2 on Neural coated culture vessel. 7. Incubate the plate at 37 C, 5% CO2. Notes: To obtain a desired cell plating density, determine the number of total viable cell using a hemocytometer and trypan blue, and dilute the cell suspension using Neural Maintenance Medium. Do not plate hccns at less than 50,000 cells/cm 2. 5

9 Maintaining Novus hccns Frozen hccns (NBP , NBP thru NBP ) 1. The morning after plating the hccns, replace the medium with fresh Neural Maintenance Medium supplemented wit Neural EnhanceTM (NBP , 1000x stock solution). 2. Culture the cells in the Neural EnhanceTM supplemented medium for two days, then switch to the Neural Maintenance Medium without Neural AdvanceTM. 3. Supplement the medium with Sure MixTM (NBP X stock solution) once every week. This prevents the clumping of the neuronal cell bodies. Plated hccns (NBP , NBP , NBP thru NBP ) 1. Open the package and place the 96-well plate into a class II biosafety cabinet. Spray the plate with 70% ethanol, and wipe down with tissue. 2. Remove the silicone sealing cap on the plate. 3. Place the plate into a 37oC, 5% CO2 incubator. Incubate the plate for 2 hours. 4. Aspirate the medium in the wells and replace with 200 μl prewarmed Neural Maintenance Medium per well of the 96-well plate. 5. Exchange medium every other day. Supplement the medium with Sure MixTM (NBP ,1000X stock solution) once every week. This prevents the clumping of the neuron cell bodies. 6

10 Technical Support Online Resources Please visit our website at for additional product information and Technical Resources, including instruction manuals, application protocols, video guides, wallcharts and webinars. Contact us For more information or technical assistance, call , or Where we are: Novus Biologicals, LLC 8100 Southpark Way, A-8 Littleton, CO USA 7