CellPlayer HUVEC NucLight Green

Transcription

1 Essen BioScience Catalog Number: 456 Storage Liquid Nitrogen Note: Cells can be thawed and cultured immediately upon receipt or stored in liquid nitrogen for long-term storage. Storage at -8 C is not recommended. Presentation 1mL, 1.7 x 1 5 cells/ml in 9% FBS, 1% DMSO Recommended Media and Components EGM-2 BulletKit (Lonza, Cat# CC-3162) 1% FBS (Cat# SH371 Thermo Hyclone) 1% Pen/Strep (Cat# 1514 Gibco/Life Technologies) CellPlayer HUVEC NucLight Green Background Each vial contains 17, Normal Human Umbilical Vein Endothelial Cells (HUVEC), pooled from multiple donors. Prior to cryopreservation, the parental HUVEC are lentivirally infected with The Essen CellPlayer NucLight Green Lentivirus (Cat# 4475) at an MOI of 6 TU/ml to express NucLight Green nuclear restricted GFP following the standard Essen protocol. This results in 7-8% transduction efficiency. A panel of validation assays designed to evaluate the effects of the nuclear label on functional cell biology was completed. These assays include cell morphology analyses, growth/proliferation, and differentiation (tube formation) compared to either parental controls or to longitudinal data collected over the past two years using the CellPlayer Angiogenesis CryoKit. Normal human cells have a limited life span in vitro. When cultured according to protocol, HUVEC NucLight Green can achieve several population doublings. Figure 1. HUVEC NucLight Green Line

2 2 % Transduced (NucLight Green) CellPlayer TM HUVEC NucLight Green Validation Assays The following experiments were completed using an IncuCyte ZOOM (1x) 1. Morphological Comparison Parental HUVEC HUVEC NucLight Green HUVEC NucLight Green Results: No significant alterations in cell morphology were observed between parental HUVEC and HUVEC NucLight Green population. 2. Proliferation Real-Time Cell Counts Using the CellPlayer Kinetic Proliferation Assay Nuclear Cnt/mm Real-Time Cell Counts of HUVEC NucLight Green cells Treated with Decreasing Serum Concentrations 1% FBS 5% FBS 2.5% FBS 1.25% FBS.63% FBS.31% FBS.16% FBS Untreated NucLight Green Transduction Efficiency at Initial and Ending Time Points Time (h) Time (hrs) Time (14hrs) Results: (Left) Real-time cell counting using the NucLight Green reagent revealed a decrease in HUVEC proliferation in medium containing decreasing amounts of serum (FBS). (Right) At both the initial time point and the assay end point, the total cell population was labeled with Vybrant DyeCycle Green to determine the percentage of the total cell population that expresses the NucLight Green nuclear label. Results illustrate a slight increase in labeled population over the course of the 14 hour assay. The slight increase may be explained by the delayed expression of detectable levels of NucLight green throughout the assay time course. Importantly, these data indicate that the labeled NucLight Green nuclear label does not negatively affect proliferation rates compared to unlabeled cells within the same culture.

3 CellPlayer TM HUVEC NucLight Green 3 3. Differentiation (Tube Formation) Kinetic Proliferation: HUVEC NucLight Green Cells in the Essen Angiogenesis Assay (at Endpoint) Kinetic Proliferation: HUVEC NucLight Green Cells in the Essen Angiogenesis Assay 15 NucLight Green Cnt/mm Tube Formation: HUVEC NucLight Green Cells in the Essen Angiogenesis Assay (CD31 label at Endpoint) Time (hrs) Cell Line Mean Tube Length ± SD CV (%) N (wells) HUVEC CytoLight Green 9.8 ± >1 HUVEC NucLight Green 12.5 ± Results: HUVEC NucLight Green cells were placed in co-culture with normal human dermal fibroblasts (NHDF) per the standard Essen BioScience CellPlayer Angiogenesis protocol in order to determine if expression of the NucLight Green nuclear label could be used to track proliferation of HUVEC in the angiogenesis assay and to determine if nuclear localization of GFP had a detrimental effect on HUVEC differentiation. The kinetic graph (above) illustrates that NucLight Green HUVEC proliferate at a constant rate in the co-culture angiogenesis assay up until approximately 2 hours (>8 days), at which time proliferation levels off. Furthermore, we show that the development of complex tube networks is not compromised by expression of nuclear restricted GFP compared to the standard HUVEC used in the CellPlayer 96-well Angiogenesis assay (Table). Related Products NucLight/CytoLight Reagents: Cat.# 4475 CellPlayer NucLight Green (Lenti, EF-1 alpha, puro) Cat.# 4481 CellPlayer CytoLight Green (Lenti, EF-1 alpha, puro) Cat.# 4513 CellPlayer CytoLight Green (Lenti, CMV, no selection) Cat.# 4476 CellPlayer NucLight Red (Lenti, EF-1 alpha, puro) Cat.# 4482 CellPlayer CytoLight Red (Lenti, EF-1 alpha, puro)

4 4 CellPlayer TM HUVEC NucLight Green NucLight Cell Lines: Cat.# 4485 CellPlayer HT-18 NucLight Red Cat.# 4487 CellPlayer MDA-MB-231 NucLight Red Cat.# 4489 CellPlayer HeLa NucLight Red Cat.# 4491 CellPlayer A549 NucLight Red Cat.# 456 CellPlayer HUVEC NucLight Green Cat.# 4453 CellPlayer HUVEC CytoLight Green Cat.# 4486 CellPlayer HT-18 NucLight Green Cat.# 4488 CellPlayer MDA-MB-231 NucLight Green Cat.# 449 CellPlayer HeLa NucLight Green Cat.# 4492 CellPlayer A549 NucLight Green Cat.# 4511 CellPlayer Neuro-2a NucLight Green Cat.# 4512 CellPlayer Neuro-2a NucLight Red Protocols and Procedures Thawing and Culturing Cells 1. The recommended seeding density HUVEC NucLight Green cells is 2,5 to 5, cells/cm 2. For example, one vial of HUVEC NucLight Green cells is sufficient to seed 1 to 3 T25 flasks. From this, calculate the number of flasks needed. 2. Prepare the flasks and culture media. 3. Remove the vial containing HUVEC NucLight Green from liquid nitrogen. 4. In a 37 C water bath, quickly thaw the vial by gentle agitation. Be careful not to submerge entire vial to avoid contamination. This process should take no more than 2 minutes. Remove vial when only a tiny ice crystal remains. 5. Wipe vial with 7% ethanol. In a biosafety hood, aliquot cells into sufficient complete media to distribute among the flasks set up in Step Gently rock the culture flasks to evenly distribute the cells and place in 37 C incubator. 7. Subculture when cells reach 7-8% confluence. For additional information on this and other products, please contact Essen BioScience at: sales@essenbio.com. Licenses and Warranty For research use only. Not for therapeutic or diagnostic use. Essen BioScience warrants that this product performs as described on the product label and in all literature associated with the sale of said product when used in accordance with the described protocol. This limited warranty is the sole warranty. No other warranties exist that extend beyond this warranty, either expressed or implied. Essen BioScience disclaims any implied warranty of merchantability or warranty of fitness for a particular purpose. Essen BioScience

5 CellPlayer TM HUVEC NucLight Green 5 disclaims any responsibility for injury or damage and shall not be liable for any proximate, incidental or consequential damages in connection with this product. If it is proven to the satisfaction of Essen BioScience that this product fails to meet performance specifications, Essen BioScience s sole obligation, at the option of Essen BioScience, is to replace the product or provide the purchaser with credit at or below the original purchase price. This limited warranty does not extend to anyone other than the purchaser. Notice of suboptimal performance must be made to Essen BioScience within 3 days of receipt of the product. This Essen BioScience product contain proprietary nucleic acid(s) coding for proprietary fluorescent protein(s) being, including its derivatives or modifications, the subject of pending patent applications and/or patents owned by Evrogen JSC (hereinafter Evrogen Fluorescent Proteins ). The purchase of Essen BioScience products incorporating these fluorescent proteins conveys to the buyer the nontransferable right to use Evrogen Fluorescent Proteins only for research conducted by the buyer. No rights are conveyed to modify or clone the gene encoding fluorescent protein contained in this product or to use Evrogen Fluorescent Proteins for commercial purposes. The right to use Evrogen Fluorescent Proteins specifically excludes the right to validate or screen compounds for commercial purposes. For information on commercial licensing, contact Evrogen Licensing Department, license@evrogen.com.