® 
        StemPro  Osteogenesis Differentiation Kit 
        Description 
                     ®
        The StemPro  Osteogenesis Differentiation Kit has been developed for the osteogenic differentiation of mesenchymal stem cells (MSCs) in tissue-culture 
        vessels. The kit contains all reagents required for inducing MSCs to be committed to the osteogenesis pathway and generate osteocytes. Using the 
                ®                                                                 ®                                ™
        StemPro  Osteogenesis Differentiation Kit in combination with StemPro  MSC SFM or MesenPRO RS  Medium provides a standardized culture 
        workflow solution for MSC isolation, expansion, and differentiation into matrix-forming osteocytes. 
                                         Product                                     Catalog no.      Amount                Storage                 Shelf life* 
                   ®
          StemPro  Osteogenesis Differentiation Kit                                  A10072-01         1 kit                   —                        — 
          Contains:                                                                                                                                       
                      ®
             StemPro  Osteocyte/Chondrocyte Differentiation Basal Medium             A10069-01        100 mL      2°C to 8°C; Protect from light   12 months
                      ®                                                                                                                            12 months 
             StemPro  Osteogenesis Supplement                                        A10066-01        10 mL        –20°C to –5°C; In the dark 
        * Shelf life duration is determined from Date of Manufacture. 
                                                                                                                                                        ®
        Product use                                                                    •  Expansion culture: Expand primary MSC isolates with StemPro  
                                                                                                                      ™
        For Research Use Only. Not for use in diagnostic procedures.                      MSC SFM or MesenPRO RS  Medium in T-75 or T-225 flasks. We 
                                                                                          have successfully tested standard growth media of DMEM+10% MSC 
        Important information                                                             Qualified FBS. We recommend refeeding the cultures every 2–3 days 
                   ®                                                                      and passaging every  
        •  StemPro  Osteogenesis Supplement is supplied frozen. Thaw prior to             5–7 days. 
          use, as described in Prepare media. 
                           ®                                                           • Passaging: We strongly recommend using low-passage MSCs (<8 
        •  Thawed StemPro  Osteogenesis Supplement is stable up to at least               to 10 passages). Continuously passaged MSCs will gradually lose 
          one month at 2°C to 8°C. Supplement can be refrozen in aliquots and             their multipotency with increased passage number (>10 passages). 
          stored at –20°C to –5°C. Avoid multiple freeze thaw cycles of                                                             ™
          supplement.                                                                  • Harvesting: We recommend using TrypLE  Express for 
                                           ®                                                                                                  ™
        •  Store prepared Complete StemPro  Osteogenesis Differentiation                  enzymatically treating and harvesting MSCs. TrypLE  Express is a 
          Medium at 2°C to 8°C in the dark. Complete medium is stable up to               recombinant protease that has been demonstrated to be gentle on 
          at least one month at 2°C to 8°C.                                               MSCs. Overexposure to trypsin will lead to reduced MSC viability 
        Safety information                                                                and expansion. 
        Read the Safety Data Sheets (SDSs) and follow the handling                     •  Timing of passaging: Do not let passaged MSCs become 
        instructions. Wear appropriate protective eyewear, clothing, and gloves.          completely confluent, as it can reduce multipotency of MSCs. 
                                                                                          Passage cultures when they reach 60–80% confluency, cell viability 
        Prepare media                                                                     is at least 90%, and the growth rate is in mid-logarithmic phase. 
        Complete Osteogenesis Differentiation Medium                                   • Seeding density: For expansion, we recommend a seeding density 
                                                                                                   3         3               2                    ™
        Thaw the supplement at 4°C, room temperature, or in a 37°C water                  of 3 × 10  to 5 × 10  viable cells/cm  with MesenPRO RS  Medium 
                                                                                                   4               2             ®
        bath, and prepare 100 mL of media according to the following table.               or 1 × 10  viable cells/cm  with StemPro  MSC SFM. 
        Store complete medium at 2°C to 8°C in the dark.                               • Confluency: Expanding MSCs in growth medium for 2–4 days 
           Osteogenesis Differentiation Medium       Concentration     Volume             before refeeding with Complete Osteogenesis Differentiation 
                   ®                                                                      Medium can enhance osteogenesis. 
          StemPro  Osteocyte/Chondrocyte                   1X           90 mL          Osteogenesis Differentiation  
             Differentiation Basal Medium                                   
                   ®                                                                                                                                       ®
          StemPro  Osteogenesis Supplement                 1X           10 mL          1.   Observe cell monolayer from basal cultures expanded in StemPro  
                                                                                                                       ™
          Gentamicin reagent (10 mg/mL)                 5 µg/mL         50 µL               MSC SFM, MesenPRO RS  medium, or standard growth medium 
                                                                                            (DMEM+10% FBS) to ensure mid-log growth phase confluence 
        MSC growth medium                                                                   (60–80%). Aspirate medium and floating cells from culture flask 
        Prepare MSC growth medium according to the following table.                         and discard. 
           MSC growth medium (500 mL)          Final concentration     Volume          2.   Add 5–10 mL DPBS. Gently rinse cell monolayer. 
          DMEM low glucose                              —              445 mL           
                                                                                                                                               ™
          MSC-qualified FBS                           10%               50 mL          3.   Remove DPBS, add 5–7 mL of pre-warmed TrypLE  Express to 
          GlutaMAX™-I (200 mM)                        2 mM              5 mL                flask and completely coat culture surface. Incubate for  
          Gentamicin reagent (10 mg/mL)              5 µg/mL           250 µL               5–8 minutes at 36°C to 38°C or until cells have fully detached. 
        Culture conditions                                                             4.   Gently pipet detached cells into a single cell solution and verify on 
        Media: StemPro® Osteogenesis Differentiation Medium                                 inverted microscope. 
        Cell line: Human mesenchymal stem cells                                        5.   Remove cell suspension from flask, transfer into a centrifuge tube, 
        Culture type: Adherent                                                              and pellet cells at 100 × g for 5–10 minutes. 
                                                                                       6.   Determine cell viability and total cell density using Trypan Blue 
                                                                          ™                 Stain and an electronic (Coulter Counter) or manual 
        Culture vessels: 12-well tissue-culture plates, 16-well CultureWell  
                                                    2                                       (hemocytometer) cell counting method. 
        slides, 96-well tissue-culture plates, or 75 cm  tissue-culture flasks 
        Temperature range: 36°C to 38°C.                                               7.   Resuspend the pellet in an appropriate volume of pre-warmed 
        Incubator atmosphere: Humidified atmosphere of 4–6% CO  in air.                     MSC Growth Medium (see Prepare media, page 1). 
                                                                    2
        Ensure proper gas exchange and minimize exposure of cultures to light.                                                      3        2
                                                                                       8.   Seed MSCs into culture vessels at 5 × 10  cells/cm . For classical 
        Important guidelines for osteogenesis differentiation                               stain differentiation assay, seed into a 12-well plate. For gene 
                                                                                            expression profile studies, seed into a T-75 flask. For 
        Publication Number MAN0000694                                                                                                               Rev. 2.00 
                  immmunocytochemisstry studies, seedd into a 16-well CultureWell™ 
                  chaambered coverglass or 96-well plate. 
           9.     Incuubate the cells inn MSC Growth MMedium at 36°CC to 38°C in a 
                  hummidified atmosphhere of 4–6% COO  for a minimumm of 2 hours up 
                  to 44 days.                                      2
           10.  Repplace media withh pre-warmed Coomplete Osteogeenesis 
                  Diffferentiation Meddium and continue incubation. MMSCs will 
                  conntinue to expand as they differenntiate under osteoogenic                                                                                                                                       
                  connditions. Refeed cultures every 33–4 days.                                                            Relaated products
           11.  Aftter specific perioods of cultivationn, you can process osteogenic                                                                        Product                                        Catalog no. 
                  culttures for alkalinee phosphatase sttaining                                                             CTS™ StemPRO® MSSC SFM                                                            A103322 
                  (7 ––14 days) or Alizzarin Red S stainning (>21 days; 
                                                                                       see the                                            ®
                  folllowing section foor method), genee expression anaalysis, or proteinn                                  StemmPRO   Human Adipose-Deriveed Stem Cell Kit                                   R7788
                  deteection.                                                                                               StemmPRO®  Adipogeenesis Differentiation Kit                                   A10070-001 
           Alizarinn Red S stain analysis                                                                                   MeseenPRO RS™ Medium                                                               12746
           1.     Aftter 21 days or lonnger under differrentiating condittion, remove                                       FBSS, MSC-Qualifiedd (non-US)                                                      12662
                  meddia from 12-well plate and rinse once with DPB                                        hh                              ™
                                                                                       S. Fix cells wit                     GlutaaMAX -I                                                                       35050
                  4% formaldehyde ssolution for 30 mminutes.                                                                Genttamicin reagentt (10 mg/mL)                                                    15710
           2.     Aftter fixation, rinsee wells twice witth distilled waterr and stain cells                                TryppLE™ Express                                                                   12604
                  withh 2% Alizarin RRed S solution (ppH 4.2) for                                                                                    ++            ++
                  2–33 minutes.                                                                                             DPBBS without Ca            and Mg                                                 14190
           3.     Rinnse wells three times with distilleed water, visualiize under light                                    Mouse anti-Osteocaalcin                                                          33-57000 
                                                                                                                                            ™
                  miccroscope and cappture images for qualitative or quuantitative                                          CultuureWell  chambbered coverglasss                                              C370055 
                  anaalysis.                                                                                                Tryppan Blue Solutionn, 0.4%                                                       15250
                                                        ®                                                                  Explanation of syymbols and waarnings 
           Imagess of cells in SttemPro  Osteeogenesis Diffferentiation 
           Mediumm                                                                                                         The syymbols present onn the product label are explained beloow: 
                                                                            ®
           Figure 1 Analysis of MSSCs cultured in SStemPro  Osteoggenesis                                                                                                           
           Differenttiation Medium ddemonstrated differentiation intoo osteogenic                                                                                                                   
           lineagess by alkaline phoosphatase staininng and Alizarin RRed S staining.                                        Tempperature Limitation    Manufacturer Batcch code             Use By: Catalog number 
                                                                                                                                                                                                                       
                                                                                                                                 CCaution, consult       Consult instructions     Keep away         Sterilized using aseptic 
                                                                                                                            accommpanying documents            for use             from light       processing techniquees 
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           Figure 22 Analysis of MSSCs cultured in SStemPro  Osteoggenesis                                                 questtiions, please conntact Life Technoologies at 
           Differenttiation Medium ddemonstrated differentiation intoo osteogenic                                          wwww.lifetechnologiees.com/support.. 
           lineagess by bone sialoprotein and osteoopontin immunosstaining. 
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