Download User Manual - Cyagen Biosciences
Transcript
User Manual OriCellTM Fischer 344(F344) Rat Mesenchymal Stem Cells with RFP (MSCs/RFP) Cat. No. RAFMX-01201 Table of Contents Contents and Storage ……………………………………………………………………………………… 3 Product Introduction ……………………………………………………………………………………… 3 Cell Characteristics and Identity ……………………………………………………………………… 3 Product Application …………………………………………………………………………………………4 General Handling Principles ……………………………………………………………………………4 Culturing OriCellTM Fischer 344(F344) Rat MSCs/RFP Thawing and Establishing OriCellTM Fischer 344(F344) Rat MSCs/RFP ……………………4 Passaging Cyagen OriCellTM Fischer 344(F344) Rat MSCs/RFP ………………………………6 Differentiation of OriCellTM Fischer 344(F344) Rat MSCs/RFP …………………………………7 Cryopreservation of OriCellTM Fischer 344(F344) Rat MSCs/RFP ……………………………11 Appendix ………………………………………………………………………………… 13 Troubleshooting ……………………………………………………………………………………………… 13 Related Products …………………………………………………………………………………………… 14 References …………………………………………………………………………………………………… 14 Technical Support ……………………………………………………………….…… 15 CONTENTS AND STORAGE Product Name Fischer 344(F344) Rat Mesenchymal Stem Cells Catalog No. RAFMX‐01201 Amount per Vial 1×106 Cells Cryopreserved At Fifth Passage Storage Condition Liquid Nitrogen CAUTION: Please handle this product as a potentially biohazardous material. This product contains dimethyl sulfoxide (DMSO), a hazardous material, in the freezing medium. PRODUCT INTRODUCTION Mesenchymal stem cells (MSCs/RFP) are multipotent stem cells that can differentiate into a variety of cell types including osteocytes, adipocytes, and chondrocytes. MSCs/RFP proliferate quickly and are capable of generating a local immunosuppressive microenvironment, thus contributing to their wide application potentials in tissue engineering, cell therapy, and gene therapy. OriCellTM Fischer 344(F344) Rat Mesenchymal Stem Cells are derived from the bone marrow of Fischer 344(F344) Rats, cultured as monolayer, and then have been transfected with a lentiviral construct containing a RFP expression motif. In addition, these cells have been tested for: • Exogenous Factors: bacterial/fungal contamination, mycoplasma contamination, and endotoxin contamination. • Characteristics: post-thaw viability, cell cycle, verification of undifferentiated state, and differentiation potential. This product is intended for laboratory research use only. It is not intended for diagnostic, therapeutic, clinical, household, or any other applications. CELL CHARACTERISTICS AND IDENTITY • Strong capacity to expand. Can be passaged at least 5 times. • Multipotent differentiation ability along the osteogenic, chondrogenic, and IMPI0075A2 RAFMX‐01201 Page 3 of 15 adipo ogenic linea ages. • Posittive for CD2 29, CD44, a and CD90 (> ( 70%), and a negativ ve for CD34 4 and CD45 5 and CD11 1b (< 5%) in flow cytometry ass says. PRODUC CT APPL LICATIO ONS Fischer 344 (F344) Rat MSCs/RFP P have beco ome a popular researcch target du ue to their otential use e in regenerative med icine and tiissue engin neering (in areas such as po ca ardiovascula ar, neural, and orthop pedic diseas se). cher 344 (F F344) Rat M MSCs/RFP can c be used d as cell mo odels to eva aluate the OriCellTM Fisc mmunoreactions, proliferation, im mmigration,, and differentiation o f MSCs/RFP P both in im viivo and in vitro. v GENERA AL HAND DLING PRINCIP P PLES h of the producct is necess sary througho out. 1.. Aseptic handling 2.. Once the e cells have been estab blished, alw ways freeze e several vi als of OriCe ellTM Fischer 344 3 (F344) Rat MSCs/ RFP as a ba ackup. Note: The O OriCellTM Fischer 344(F F344) Rat MSCs/RFP M can c be froze en/thawed d at least N tw wo times. ecommende ed to use ce ells that are e at, or und der, an 3.. For all studies, it is strongly re p number of 10 0. original passage 4.. For general mainten nance of ce ells, we reco ommend th he seeding density to be 2.02 c . 3.0×104cells/cm 5.. For general mainten nance of ce ells, we reco ommend th hat the med dium is cha anged if it e pH indicattor in the medium m app pears yellow w). In general, becomes acidic (the very three days. d change the growth medium ev 6.. Do not le et OriCellTM Fischer 344 4(F344) Ra at MSCs/RF FP overgrow w as it will result r in contact in nhibition. When W the ce ells are 80--90% confluent, subcu ulturing the e cells is strongly recommend ded. Note: We sttrongly reco ommend th he use of Or riCellTM cult ture media and other related N re eagents for optimal re esults. THAWIN NG AND D ESTABLISHIN G OriCe ellTM FIS SCHER 3 344(F34 44) RAT T MSCs/R RFP M Materials Required R • OriCellTM Mesenchym mal Stem C Cell Growth Medium (C Cat. No. GU UXMX-9001 11) IMPI0075A2 RA AFMX‐01201 Page 4 of 15 Th hawing and a Estab blishing F ischer 34 44(F344) Rat MSC s/RFP m the fully supplemen s ted (complete) OriCelllTM MSC Grrowth Medium to 1.. Pre-warm 37°C. 2.. Add 9 mL L of OriCellTM MSC Gro owth Mediu um to a 15 mL conical tube. 3.. Remove the cryovia al of OriCelllTM Fischer 344(F344) Rat MSCs//RFP from liquid nitrogen.. 4.. Quickly thaw t the cryovial in a 37°C water bath until the last icce crystal disappears. For optim mal results, be sure to o finish the thawing prrocedure wiithin 3 minutes. Be careful not to subm merge the en ntire vial. Maximum M cell viability y is depende ent on the d complete thawing off frozen cells. rapid and ess than op ptimal if the e cells are thawed t forr more than n 3 minutes s. N Note: Resultts will be le a the cells s are complletely thawed, disinfec ct the outsiide of the cryovial c 5.. As soon as with 70% % v/v ethan nol. TM 6.. Use a pip pette to transfer the ccells to the 15 mL coniical tube co ontaining OriCell O MSC Growth Medium m inside a biosafety cabinet. c Be careful nott to introduce any d the transfer prrocess. bubbles during 7.. Rinse the e vial with 1 mL of the e medium to t reduce cell loss. Su bsequently y transfer this 1 mL L of cell sus spension in nto the conical tube. 8.. Gently mix m the cell suspension n by slowly pipetting up u and dow wn. Be carefful not to introduce e any bubbles. 9.. Centrifug ge the cell suspension s at 250 x g for 5 minu utes. 10 0. Carefully y aspirate off as much of the supernatant as s possible a and add 2-3 3 mL of fresh OriCellTM MSC Growth Me edium (pre-warmed to o 37°C). 11 1. Gently re esuspend th he cells in O OriCellTM MS SC Growth Medium. 12 2. Seed the e cells into a T25 flask k and add a sufficient amount a of OriCellTM MSC M Growth h Medium. Gently roc ck the cultu ure flask to o evenly dis stribute the e cells. 13 3. Incubate e the flask at a 37°C ins ide a 5% CO C 2 humidiffied incubattor. 14 4. The nextt day, chang ge the med dium with fresh f growth medium (pre-warmed to 37°C C). 15 5. Change the t growth medium ev very three days thereafter. 16 6. When the e cells are approximattely 80-90% % confluent, they can n be dissociated with Trypsin-E EDTA and passaged. p Note: Chang ging Mediium N 1.. Warm an n appropriatte amount of medium to 37°C in a sterile co ontainer. Replace R the e spent me edium with the pre-wa armed, fres sh medium.. Once com mpleted, retturn the flask to the t incubator. 2.. Avoid rep peated war rming and c cooling of the medium m. If the en ntire conten nt is not needed fo or a single procedure,, transfer only o the req quired volu me to a ste erile secondarry container. IMPI0075A2 RA AFMX‐01201 Page 5 of 15 Fig. 1. OriCellTM Fischer 344(F344) Rat Mesenchymal Stem Cells with RFP are established. PASSAGING OriCellTM FISCHER 344(F344) RAT MSCs/RFP Materials Required • 0.25%Trypsin-0.04%EDTA (Cat. No. TEDTA-10001) • Phosphate-Buffered Saline (1×PBS) (Cat. No. PBS-10001) • OriCellTM Fischer 344(F344) Rat Mesenchymal Stem Cells with RFP (Cat. No. RAFMX01201) • OriCellTM Mesenchymal Stem Cell Growth Medium (Cat. No. GUXMX-90011) Passaging OriCellTM Fischer 344(F344) Rat MSCs/RFP 1. Pre-warm the OriCellTM MSC Growth Medium, 1×PBS, and Trypsin-EDTA solution to 37°C. 2. Carefully aspirate the spent medium from the 80-90% confluent monolayer of MSCs/RFP. 3. Add 1×PBS (6 mL for T75 flask, 3 mL for T25 flask). Be careful not to disturb the monolayer. Gently rock the flask back and forth to rinse the monolayer. 4. Aspirate 1×PBS off and discard. 5. Repeat steps 3-4 two or three times. 6. Add 0.25%Trypsin-0.04% solution (2-3 mL for T75 flask, 1 mL for T25 flask). Gently rock the flask back and forth to ensure that the entire monolayer is covered with the Trypsin-EDTA solution. Allow trypsinization to continue until the majority of the cells (approximately 80%) are rounded up. At this point, gently tap the side of the flask to release the majority of cells from the culture flask surface. Important: Avoid leaving cells exposed to the trypsin longer than necessary (no more than two minutes if using Cyagen’s trypsin-EDTA solution). Care should also be taken that the cells are not forced to detach prematurely as this may result in clumping. 7. After the cells are visibly detached, immediately add the pre-warmed OriCellTM MSC IMPI0075A2 RAFMX‐01201 Page 6 of 15 Growth Medium M (6 mL for T75 5 flask, 3 mL for T25 flask) to neu utralize the e trypsiniza ation. 8.. Gently piipette the medium m ove er the cells to dislodge e and resusspend the cells. c Repeat 5-6 times un ntil all the ccells are dis ssociated frrom the fla ask and eve enly d into a single cell susspension. dispersed 9.. Transfer the dissocia ated cells in nto a 15 mL m conical tu ube. 10 0. Centrifug ge at 250 x g for 5 min nutes. 11 1. Carefully aspirate off as much of the supe ernatant as s possible. T MSC Gro 12 2. Add 2 mL L of OriCellTM owth Mediu um to the co onical tube e and gently y resuspend d the cells thoroughly y. 13 3. Plate the cells into appropriate a e flasks. OriCellTM Fisc cher 344(F3 344) Rat MSCs/RFP plit at 1:2 or o other ap propriate ratios. can be sp 14 4. Add an appropriate amount off medium to o the cells. Incubate tthe cells att 37°C 5 CO2 hu umidified in cubator. inside a 5% N Note: Care should be taken t to av void introdu ucing bubbles during pipetting. Additional Tips Tiime to Cha ange Mediium It is recomm mended to change c the culture me edium if the ere are too many dead d cells afterr pa assaging. It is recomm mended to change c the culture me edium when never the m medium bec comes ac cidic, even if the cells do not rea ch 80-90% % confluency y. The pH iindicator in the culture e m medium will appear yellow when a acidic. bculture Tiime to Sub W When OriCelllTM Fischer 344(F344)) Rat MSCs//RFP are 80-90% con nfluent, it is i re ecommende ed that the e cells be su ubcultured.. Do not lett the cells o overgrow as s it will re esult in contact inhibition. OriCellTTM FISCH HER 344 4(F344) RAT MS SC DIFFE ERENTIA ATION USING U T TM OriCell DIFFE ERENTIA ATION M MEDIA cher 344(F3 344) Rat M MSCs/RFP ca an differenttiate into a variety of cell types OriCellTM Fisc dipocytes, and chondrrocytes. including ostteocytes, ad O Osteogenic Differen ntiation M Materials Required R senchymal Stem Cell O Osteogenic c Differentia ation Mediu um (Cat. No o. GUXMXOriCellTM Mes 0021) 90 IMPI0075A2 RA AFMX‐01201 Page 7 of 15 sis Protoco ol Osteogenes Note: The protocol listed below iss for 6-welll tissue cultture platess. N t OriCellTM Fischer 3 44(F344) Rat R MSCs/R RFP in OriCe ellTM Mesen nchymal 1.. Culture the Stem Cell Growth Medium M at 3 37°C in a 5% 5 CO2 hum midified inccubator. 2.. When cellls are apprroximately 80-90% co onfluent, they can be d d with dissociated 0.25%Try ypsin-0.04%EDTA (Ca at. No. TED DTA-10001) ). 2 3.. Reseed the MSCs in n the growt h medium at 3×104 cells/cm c in a 6-well tissue oated with 0 0.1% gelatin solution. culture plate pre-co 4.. Incubate the cells at 37°C insi de a 5% CO2 humidifiied incubattor. 5.. When cellls are apprroximately 60-70% co onfluent, ca arefully asp pirate off the growth medium from each well and ad dd 2 mL of OriCellTM Mesenchyma M ell al Stem Ce nic Differentiation Med dium. Osteogen 6.. Feed cells every thrree days forr 2-4 weeks by completely replaccing the medium with h CellTM Mese enchymal S Stem Cell Osteogenic O Differentiat D tion Medium m (prefresh OriC warmed to t 37°C). 7.. After 2-4 4 weeks of differentiat d ion, cells ca an be fixed and staine ed with aliz zarin red S. event osteoblasts from m detaching g, it is recommended tto change half h of the ote: To pre No me edium everry two days s before ana alysis. d S Stainin ng Analysiis Allizarin Red ve the osteo ogenic diffe erentiation medium 1.. After the cells have differentia ted, remov from the wells and rinse with 1 1x phospha ate-buffered d saline (PB BS). Fix ce ells with 2 mL of 4% % formaldeh hyde soluti on for 30 minutes. m 2.. Rinse we ells twice wiith 1x PBS. Stain the cells with 1 mL alizarrin red S wo orking solution for f 3-5 min nutes. 3.. Rinse we ells 2-3 time es with 1x PBS. 4.. Cells can now be vis sualized an nd analyzed d under a microscope. m IMPI0075A2 RA AFMX‐01201 Page 8 of 15 Fig g. 3 OriCellTM Fischer 344(F F344) Rat MSCs/RFP are differentiated in nto osteocytes and a are stained wiith alizarin red d S. Adipogenic Differen ntiation M Materials Required R OriCellTM Mes senchymal Stem Cell A Adipogenic c Differentia ation Mediu um (Cat. No o. GUXMX90 0031) Ad dipogenes sis Protoco ol Note: The protocol listed below iss for 6-welll tissue cultture platess. N 1. Culture the t OriCellTM Fischer 3 44(F344) Rat R MSCs/R RFP in the O OriCellTM Me esenchymal Stem Cell Growth Medium M at 3 37°C in a 5% 5 CO2 hum midified inccubator. onfluent, they can be d d with dissociated 2.. When cellls are apprroximately 80-90% co 0.25%Try ypsin-0.04%EDTA (Ca at. No. TED DTA-1000). 3.. Reseed the MSCs in n growth me edium at 2x104 cells/c cm2 in a 6--well tissue culture h a medium m volume o of 2 mL per well. plate with 4.. Incubate the cells at 37°C in a 5% CO2 humidified h incubator. 5.. Feed the cells every y three day ys until they y are 100% % confluent or post-confluent. Induction n of adipoge enic differe entiation at post-confluency is strrongly reco ommended.. 6.. When the e cells are 100% conffluent or po ost-confluen nt, carefully y aspirate off o the spent gro owth mediu um from the e wells and d add 2 mL of OriCellTMM Mesenchy ymal Stem Cell Adipogenic Differentiation medium A (induction medium) per well. 7.. Three days later, ch hange the m medium to OriCellTM Mesenchyma M al Stem Cell nic Differentiation med dium B (ma aintenance medium) b by complete ely Adipogen replacing g the spent medium A . 8.. 24 hours later, chan nge the me edium back to MSC Ad dipogenic D Differentiatio on medium m A. 9.. To optimally differentiate MSC Cs into adipo ogenic cells s, repeat th he cycle of induction ntenance att least three e times. and main M 10 0. After thre ee to five cycles of ind duction and d maintenance, culture e the cells in OriCellTM Mesenchy ymal Stem Cell Adipog genic Differentiation medium m B ffor an addittional 4-7 days until the lipid droplets d are e big, round enough. During the se days period, m every thre ee days. change the medium Oil Red O Stain S Analy ysis e cells have differentia ated, remov ve the MSC maintenan nce medium m from the 1.. After the wells and d rinse with h 1x phosph hate-bufferred saline (PBS). Fix ccells with 2 mL of 4% formalde ehyde solution for 30 m minutes. 2.. Rinse we ells twice with 1x PBS and stain cells c with 1 mL of oil rred O workiing solution n IMPI0075A2 RA AFMX‐01201 Page 9 of 15 (3:2 dilution with distilled water and filter with filter paper) for 30 minutes. 3. Rinse wells 2-3 times with 1x PBS. 4. Cells can now be visualized and analyzed under a microscope. TM Fig.4 OriCell Fischer 344(F344) Rat MSCs/RFP are differentiated into adipocytes and are stained with oil red O. Chondrogenic Differentiation Materials Required OriCellTM Mesenchymal Stem Cell Chondrogenic Differentiation Medium (Cat. No. GUXMX-90041) Chondrogenesis Protocol 1. Calculate the total number of MSC pellet cultures required for your experiment (2.5×105 MSCs/RFP are needed to form each chondrogenic pellet). Transfer this amount of cells into an appropriate culture tube. 2. Wash the MSCs/RFP with Incomplete Chondrogenic Medium. Centrifuge the cells at 150 x g for 5 minutes at room temperature and then aspirate off the supernatant. Resuspend the cells in 1 mL of Incomplete Chondrogenic Medium per 7.5×105 cells. Centrifuge again at 150 x g for 5 minutes and then aspirate off the medium. 3. Resuspend the MSCs/RFP in Complete Chondrogenic medium to a concentration of 5.0×105 cells/mL. 4. Aliquot 0.5 mL (2.5×105 cells) of the cell suspension into 15 mL polypropylene culture tubes. Centrifuge the cells at 150 x g for 5 minutes at room temperature. DO NOT aspirate the supernatant or resuspend the pellet. 5. Loosen the caps of the tubes one half turn in order to allow gas exchange, and incubate the tubes at 37°C in a humidified atmosphere of 5% CO2. Do not disturb the pellets for 24 hours. 6. Feed the cell pellets every 2-3 days by completely replacing the medium in each tube (to avoid aspirating the pellets when aspirating the medium, attach a sterile 1200μL pipette tip to the end of the aspirating pipette). Add 0.5 mL of freshly prepared Complete Chondrogenic Medium to each tube. IMPI0075A2 RAFMX‐01201 Page 10 of 15 7. After replacing the medium, flick the bottom of the tube to ensure that the pellet is free floating. Loosen the caps and return the tubes to the 37°C incubator. 8. Chondrogenic pellets should be harvested after 14-28 days in culture. Pellets may be formalin-fixed and paraffin-embedded for alcian blue stain analysis. Alcian Blue Staining Procedure 1. The tissue sample should be formalin-fixed and paraffin-embedded already. 2. Staining procedure: a) Deparaffinize slides and hydrate to distilled water. b) Stain in alcian blue solution for 30 minutes. c) Wash in running tap water for 2 minutes. d) Rinse in distilled water. e) Visualize under a light microscope and capture images for analysis. Blue staining indicates synthesis of proteoglycans by chondrocytes. TM Fig.5 OriCell Fischer 344(F344) Rat MSCs/RFP are differentiated into cartilages and are stained with alcian blue. IMPI0075A2 RAFMX‐01201 Page 11 of 15 CRYOPR RESERVA ATION OF O CELL LS USIN NG OriCe ellTM CRYOPR RESERVA ATION MEDIA M R Protein-F Free Cryoprreservation Medium (C Cat. No. NC CPF-10001) is a OriCellTM NCR use freezing g medium. Its chemically-define ed and prottein-free prrotein-free,, ready-to-u fo ormulation has h been optimized to o stem cells s and prima ary cells, th hus greatly enhancing th he viability and integrity of these cells by prrotecting th hem from da amage durring the on ne-step free eze-thaw procedure. p Unlike other conventiional freezi ng media, which re equire a slow programmed freeze e, this prod duct allows the cells to o be directly y frozen at 80 0°C. Cryopreservation ge the cultu ure medium m with fresh h growth medium m 24 h hours before freezing Note: Chang g. N ells that are e in the log garithmic growth phas se. Perform m a cell count to 1.. Collect ce determin ne the viable cell dens ity. 2.. Centrifug ge the cells for 3-5 mi nutes at 25 50 x g and 20°C. Rem move and discard the supernattant using a pipette. 3.. Resuspen nd the cell pellet in th e OriCellTM NCR Protein-Free Cry yopreservation Medium m at a cell density d of 10 1 5-106 cellls/mL. 4.. Dispense e aliquots of the cell su uspension into cryogenic storage e vials that are properly labeled. 5.. Place the e vials direc ctly in a -80 0°C freezerr. After 24 hours, tran nsfer the fro ozen vials to liquid nitrogen fo or long-term m preservattion. IMPI0075A2 RA AFMX‐01201 Page 12 of 15 APPENDIX Troubleshooting The table below lists some potential problems and solutions for culturing MSCs/RFP. Problem Low cell recovery rate Cause Solution The storage condition does not meet the requirements Purchase a replacement and store in liquid nitrogen for long‐term preservation. Thawing of the cells takes too long Thaw cells for no more than 3 minutes. Cells are incompletely recovered after thawing After aspirating off medium, wash the tube with culture medium twice and transfer all of the cells to the dish. Cells are handled roughly Care should be taken to avoid introducing bubbles during pipetting. Also avoid vortexing and high‐speed centrifugation Medium is not pre‐warmed Warm medium to 37°C before recovery. Mycoplasma contamination Discard the cells in question and disinfect the laboratory environment before recovering the next batch of cells. Slow cell growth Over digestion Wash the cells with PBS 2‐3 times to remove serum prior to trypsinization (serum will inhibit the function of trypsin). Control the digestion time. Plating density is too low Increase the plating density. Inappropriate serum and medium Use Cyagen tailor‐made culture media. If other serum and media products are used, please perform validation to ensure compatibility. Dead cells are not removed promptly Change the medium next day after recovery to ensure removal of all dead cells. Cell Contamination Discard the cells in question and disinfect the laboratory environment before recovering the next batch of cells. Plating density is too low Some stem cells can secrete factors to support cell growth. Therefore, a certain degree of plating density must be maintained; otherwise, it will lead to cell proliferation slow down and cell aging. Cell aging IMPI0075A2 RAFMX‐01201 Page 13 of 15 Over digestion Cell aging Wash the cells with PBS 2‐3 times to remove serum prior to trypsinization (serum will inhibit the function of trypsin). Cells show spontaneous differentiation Ineffective induction of cell differentiation Control the digestion time. The passaging time is not appropriate The cells should be subcultured when reaching 80‐90% confluency in order to avoid contact inhibition. DMSO is not completely removed during cell recovery Wash the cells with pre‐warmed medium 2‐3 times during recovery. Differentiation reagents need to be optimized Cell passage is too high Use Cyagen tailor‐made differentiation media. Use cells at a low original passage number. RELATED PRODUCTS Product Catalog Number OriCellTM Mesenchymal Stem Cell Growth Medium GUXMX-90011 OriCellTM Mesenchymal Stem Cell Osteogenic Differentiation Medium GUXMX-90021 OriCellTM Mesenchymal Stem Cell Adipogenic Differentiation Medium GUXMX-90031 OriCellTM Mesenchymal Stem Cell Chondrogenic Differentiation Medium GUXMX-90041 0.25%Trypsin-0.04%EDTA TEDTA-10001 Phosphate-Buffered Saline (1xPBS) PBS-10001 OriCellTM NCR Protein-Free Cryopreservation Medium NCPF-10001 REFERENCES Jiang, Yuehua, Jahagirdar, Balkrishna N, and Reinhardt, R Lee.(2002)Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418:41-49. Hideya Yoshimura, Takeshi Muneta, and Akimoto Nimura. (2006)Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell and Tissue Research 327:449-462. IMPI0075A2 RAFMX‐01201 Page 14 of 15 Cyagen Biosciences reserves all rights on the technical documents of its OriCellTM cell culture products. No part of this document may be reproduced or adapted for other purposes without written permission from Cyagen Biosciences. IMPI0075A2 RAFMX‐01201 Page 15 of 15