Name of Products:GFP Expressing Human Umbilical Vein Endothelial Cells (GFP-HUVECs) 

 Catalogue Number:cAP-0001GFP

 Product Format:Frozen Vial

 Cell Number:>5x10[5] cells/vial

 GENERAL INFORMATION

 HUVECs (cAP-0001) were infected with GFP- expressing Lentiviral particles @ passage 1. Puromycin resistant GFP-HUVECs (cAP-0001GFP) were selected and

 shipped in frozen vials (cells are provided @ passage 3). Endothelial Growth Medium (cAP-02) is recommended for cell culture and these cells have a minimum of 18

 population doubling levels when cultured following the protocols described below).

 CHARACTERIZATION OF THE CELLS

 1. Cytoplasmic VWF/Factor VIII >95% positive by immunofluorescence

 2. Cytoplasmic uptake of Di-I-Ac-LDL >95% positive by immunofluorescence

 3. Cytoplasmic PECAM1 >95% positive by immunofluorescence

 4. HUVECs are negative for HIV-1, HBV, HCV, and mycoplasma

 PRODUCT USAGE

 Cells are offered for Research Use Only.

 SHIPPING

 Frozen Vials in Dry Ice package.

 HANDLING OF ARRIVING CELLS

 When you receive the dry ice package with cells in frozen vials, transfer the frozen vials of cells into a -80C freezer for short period storage or a liquid nitrogen tank for

 long- term storage.

 PROTOCOLS FOR THAWING THE CELLS AND SUBCULTURE

 A. Pre-coating of T25 flasks- Add 2ml each Quick Coating Solution (cAP-01) into a T25 flask to cover the whole surface of the flask, 5 mins

 later, dispose the excessive coating solution by aspiration and the flask is ready to be used (although solution containing other extracellular

 matrix, i.e. gelatin, collagen, and fibronectin, can be used, make sure to optimize the conditions in advance).

B.  Thaw the frozen cell vial in a 37C water bath first, and then transfer the cells into the pre-coated T25 flask with 10ml of cAP-02 medium,

 cells usually become confluent overnight and ready to be passaged.

C. To passage the cells, rinse the cells in a T25 flask with 5ml HBSS (RT) twice; then add 2ml Trypsin/EDTA (RT) (cAP-23) into one T25 flask;

 gently dispose the excessive Trypsin/EDTA solution within 20 seconds by aspiration.

 D. Leave the T25 flask with the cells at RT or 37C for 1 min (most cells usually will detach from the surface within 1-2 mins; or monitor the

 cells under a microscope until most of cells become rounded up, and then gently tap the flask against the bench surface, and the cells will

 move on the surface of the flask when monitoring under microscope.

 E.Add 5ml Trypsin Neutralization Buffer and spin down the cells with 800RPM centrifugation for 5 mins.

 F.Re-suspend the cell pellet with 10 or 15ml cAP-2 medium and transfer 5 ml each into 2 or 3 pre-coated T25 flasks (for 1/2 to 1/3

 subculture ratio).

 G.Change medium every 2 or 3days and the cells usually become confluent within 7 days (when split at a 1/3 ratio).

 H.To prepare quiescent cells, when cells are nearly confluent, replace cAP-02 with Endothelial Basal Medium (EBM, cAP-03) containing

 0.5%FBS for about 8-12hrs before your experiments

 Name of Products:RFP Expressing Human Umbilical Vein Endothelial Cells (RFP-HUVECs)

 Catalogue Number:cAP-0001RFP

 Product Format:Frozen Vial

 Cell Number:>5x10[5] cells/vial

 GENERAL INFORMATION

 HUVECs (cAP-0001) were infected with RFP- expressing Lentiviral particles @ passage 1. Zeocin resistant RPF-HUVECs (cAP-0001RFP) were selected and shipped

 in frozen vials (cells are provided @ passage 3). Endothelial Growth Medium (cAP-02) is recommended for cell culture and these cells have a minimum of 18 population

 doubling levels when cultured following the protocols described below)

 CHARACTERIZATION OF THE CELLS

 1. Cytoplasmic VWF/Factor VIII >95% positive by immunofluorescence

 2. Cytoplasmic uptake of Di-I-Ac-LDL >95% positive by immunofluorescence

 3. Cytoplasmic PECAM1 >95% positive by immunofluorescence

 4. HUVECs are negative for HIV-1, HBV, HCV, and mycoplasm

 PRODUCT USAGE:Cells are offered for Research Use Only.

 SHIPPING:Frozen Vials in Dry Ice package.

 HANDLING OF ARRIVING CELLS:When you receive the dry ice package with cells in frozen vials, transfer the frozen vials of cells into a -80C freezer for short period storage or a liquid nitrogen tank for long- term storage.

 PROTOCOLS FOR THAWING THE CELLS AND SUBCULTURE

A. Pre-coating of T25 flasks- Add 2ml each Quick Coating Solution (cAP-01) into a T25 flask to cover the whole surface of the flask, 5 mins

 later, dispose the excessive coating solution by aspiration and the flask is ready to be used (although solution containing other extracellular

 matrix, i.e. gelatin, collagen, and fibronectin, can be used, make sure to optimize the conditions in advance).

 B.Thaw the frozen cell vial in a 37C water bath first, and then transfer the cells into the pre-coated T25 flask with 10ml of cAP-02 medium,

 cells usually become confluent overnight and ready to be passaged.

 C.To passage the cells, rinse the cells in a T25 flask with 5ml HBSS (RT) twice; then add 2ml Trypsin/EDTA (RT) (cAP-23) into one T25 flask;

 gently dispose the excessive Trypsin/EDTA solution within 20 seconds by aspiration.

D. Leave the T25 flask with the cells at RT or 37C for 1 min (most cells usually will detach from the surface within 1-2 mins; or monitor the

 cells under a microscope until most of cells become rounded up, and then gently tap the flask against the bench surface, and the cells will

 move on the surface of the flask when monitoring under microscope.

 E.Add 5ml Trypsin Neutralization Buffer and spin down the cells with 800RMP centrifugation for 5 mins.

F. Re-suspend the cell pellet with 10 or 15ml cAP-2 medium and transfer 5 ml each into 2 or 3 pre-coated T25 flasks (for 1/2 to 1/3

 subculture ratio).

 G.Change medium every 2 or 3days and the cells usually become confluent within 7 days (when split at a 1/3 ratio).

 H.To prepare quiescent cells, when cells are nearly confluent, replace cAP-02 with Endothelial Basal Medium (EBM, cAP-03) containing

 0.5%FBS for about 8-12hrs before your experiments.

 Name of Product:RFP-Human Lung Fibroblasts (HLFCs)-Adults (RFP-HLFCs-ad)

 Catalog Number:cAP-0033RFP

 Product Format:Frozen Vial

 Cell Number:>5x10[5]cells/vial

 GENERAL INFORMATION

 HLFCs-Ad are isolated from healthy human lung tissue (adult) samples and RFP-Human Lung Fibroblasts (RFP-HLFCs-ad) were selected by RFP-lentiviral infected

 human lung fibroblasts with 4ug/ml of Zeocin and shipped in frozen vials (the cells are provided @ passage 3). Fibroblast Growth Medium (FGM) (cAP-44) is

 recommended for cell culture and these cells have an average minimum population doubling levels > at least 10 when cultured following the detailed protocol described

 below). HLFCs-Ad are tested negative for HIV-1, HBV, HCV, and mycoplasma.

 Product Use RFP-HLFCs-Ad are for research use only.

 Shipping Frozen Vial

HANDLING OF ARRIVING CELLS

 When you receive the cells in a frozen vial, you can transfer the vial of cells into a -80C freezer for short term storage or a liquid nitrogen tank for long term storage.

 Thaw the cells in a 37C water bath, and then transfer the cells into a T25 flask pre-coated with Quick coating solution (cAP-01) as described in detail in Subculture

 Protocol

 SUBCULTURE PROTOCOL

 A) Pre-coating of T25 flasks- Add 2ml of Quick Coating Solution (cAP-01) into one T25 flask and make sure whole surface of the flask is

 covered with the coating solution. Five minutes later, dispose excessive Quick Coating Solution by aspiration and the flask is ready to be

 used (no need for overnight incubation when using Quick Coating Solution). Other extracellular matrix can be used including gelatin,

 collagen, and fibronectin and you are advised to test the conditions for using those materials in advance.

 B) Rinse the cells in T25 flask with 5ml HBSS (Room Temperature, RT) twice.

 C) Add 2ml of Trypsin/EDTA (RT) (cAP-23) into one T25 flask (make sure the whole surface of the T25 flask is covered with Trypsin/EDTA),

 and gently dispose the excessive Trypsin/EDTA solution within 20 seconds with aspiration.

 D) Leave the T25 flask with the cells at RT for 1 minute (the cells usually will detach from the surface within 1-2 minutes). You can monitor

 the cells under the microscope and when most of cells become rounded up, hit the flask against the bench surface, and the cells will move

 on the surface of the flask when monitoring under a microscope.

 E)Add 5ml Trypsin Neutralization Buffer (cAP-28) and spin the cells down with 800RPM for 5 minutes.

 F) Re-suspend the cell pellet with 10-15ml of full medium and the cell suspension is transferred directly into 2 or 3 pre-coated T25 flasks (5ml

 each, and the cells are sub-cultured at 1

G)Change medium every 2-3 days and cells usually become confluent within 7 days (when split at a 1)