Name: analysis of minerals produced by hfob 1.19 and saos-2 cells using transmission electron microscopy with energy dispersive x-ray microanalysis
Uploaded: 2018-06-24T13:00:00
Description: Watch this Scientific Journal Video about Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis at JoVE.com

MK og Spørg udføres manuelle operationer og LB udarbejdet tegninger og lavede filmen. Spørg skrev manuskriptet, LB skrev manuskriptet og MK forberedt tabellen. SM, RB og SP læse kritisk tabellen, scriptet og manuskriptet. Forfatterne vil gerne takke Hanna Chomontowska for hendes fremragende hjælp med ultramikrotomi Szymon Suski og Henryk Bilski for deres fremragende hjælp med TEM-EDX analyse. Forfatterne vil gerne takke Dr. Patrick Lunde til professionel engelsk korrektion og Barbara Sobiak til optagelse af vejledningen.
Dette arbejde blev støttet af tilskud Nielsen N401 140639 fra det polske ministerium for videnskab og videregående uddannelse med ASK, af tilskud fra National Science Centre, Polen 2016/23/Nielsen/NZ4/03313 til LB og 2016/23/Nielsen/NZ1/02449 til MK, EU RP7 projekt BIOIMAGINE : BIO-IMAGing forskning INnovation og uddannelse, GA No. 264173, og af de lovpligtige midler af Nencki Institut for eksperimentel biologi, Polish Academy of Sciences.

1. celle kultur og behandling
<ol><li>Sætte alle de nødvendige materialer under laminar flow hood og sterilisere dem under UV-lys. Kultur medier er: en 1:1 blanding af skinke er F12 og DMEM medier med 2,5 mM L-glutamin suppleret med 100 U/mL penicillin, 100 U/mL streptomycin, 0,3 mg/mL G418 og 10% føtal bovint Serum (FBS) (v/v) for menneskelige fosteret hFOB 1,19 SV40 store T antigen transfekteret osteoblaster og Mccoy's 5A medium med 1,5 mM L-glutamin supplere med 100 U/mL penicillin, 100 U/mL streptomycin og 15% F...

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I den nuværende papir, vi beskrevet protokollerne for AR-S farvning, UV lys identifikation af fluorapatite og TEM-EDX in vitro- imaging af MVs udgivet af mineralizing celler og mineraler produceret af MVs. Det er muligt at behandle alle metoder nævnt ovenfor ved at følge nogle fælles fejlfindingstrin. For at opnå optimale resultater, skal flere kritiske trin udføres omhyggeligt. For det første er det bedre at tilføje AA (som er sure) efterfulgt af β-GP (som er basisk) for at bevare pH 7,4 af n?...

Bone er en type af bindevæv består af to dele: økologisk (celler og kollagen fibre) og mineraler (calcium og fosfat forbindelser). De vigtigste mineralske bestanddele i knogler er apatit1. Forskellige typer af mineralisering-kompetente celler i knoglen (osteoblaster), tænder (odontoblasts) og brusk (chondrocytter) regulere de indledende trin i mineralisering af producerer proteiner i den ekstracellulære matrix (ECM) og frigive matrix vesikler (MVs) (figur 1). MVs er 100-300 nm diameter blærer, der ophobes calcium og fosfat lette apatit Nukleering og efterfølgende bindes til kollagen2,3. Derefter, MVs smuldre for at frigive apatit til den ekstracellulære medium. Apatit fortsætter med at vokse i kontakt med kollagen fibre og danne knoglematrix. Mineralisering er påført den konstant tilførsel af P,jeg og Ca2 + i den ekstracellulære medium. Nogle nyligt offentliggjorte data understøtter vores model4,5. Bløde væv ikke mineralize fysiologiske betingelser. Ektopisk kalcifikation kan dog forekomme patologiske betingelser såsom vaskulære forkalkning3. Vaskulære celler, at erhverver osteoblastdannelse Fænotypen kan producere MVs, der fremkalde Nukleering af apatit og indlede mineralisering i de mediale og intima lag i væggen af blodkar. Siden ektopisk kalcifikation ligner normale endochondral mineralisering3, forstå de molekylære mekanismer af mineralisering af ossøse celler og chondrocytter bør give nogle fingerpeg om ektopisk kalcifikation af bløde væv, der er dannet.
Udviklingen af skeletmuskulatur væv er reguleret af forskellige enzymer, vækstfaktorer, og projektledere eller hæmmere af mineralisering. Handlingen antagonistiske væv-uspecifik alkalisk fosfatase (TNAP) (figur 1) og ectonucleotide pyrophosphatase/phosphodiesterase jeg (NPP1), sammen med ankyrin (ANK), kontrollerer uorganiske pyrofosfat, (PPjeg) koncentration 6. PPjeg, en potent hæmmer af dannelse af HA er hydrolyseret af TNAP; NPP1 hydrolyserer nukleotid triphosphates for at danne PP,jeg mens ANK eksport PPjeg fra cellen til ECM. Pi/PPi forholdet kan regulere apatit dannelse7,8 med mulige patologiske konsekvenser9.
MV-membranen er beriget med ion transport proteiner, som letter den indledende udfældning af calcium og fosfat inde MVs under Nukleering processen (figur 1). Fosfat transporter 1 (PiT) hjælper med at indarbejde Pjeg genereret i perivesicular rum i MVs10,11. Annexins kan være involveret i bindingen og transport af Ca2 + og den biofysiske proces, der initierer mineralisering i MV lumen12,13. Vi favoriserer den hypotese, foreslog tidligere, for mineralisering inden intracytoplasmatisk blærer af indre Nukleering af apatit inde MV før dens formering i ECM14,15. In vitro modellering bekræftet induktion af Ca2 +/Pjeg komplekser dannelse i proteoliposomes af PS og AnxA516. Dette kan indikere at ophobning af Ca2 +, P,jeg, AnxA5 og PS komplekser i lipid rafts i microvilli-lignende membranesrepresent Nukleering core (NC) af apatit inden for Mvs Annexins og TNAP også besidder kollagen-bindende kapacitet, som kan være nyttige i at placere MVs langs kollagen fibre og stimulere udbredelsen af mineralisering i ECM. Fetuin A og osteopontin (OPN)17, er kendt som hæmmere af apatit formation, som kan bremse udbredelsen af mineralisering på kollagen skafottet. Nukleering og formering er forskellige begivenheder, den tidligere forud for de sidstnævnte, og begge kan være relevante for processen med patologisk mineralisering.
For at opdage, hvordan kemien i calciumphosphat komplekser kan ændre fysiologiske mineralisering og ektopisk kalcifikation, er det nødvendigt at identificere de mineraler, der er produceret af celler. Apatit er en gruppe af calcium og fosfat indeholder mineraler med generelle krystal enhed celle formel Ca10(PO4)6X2, hvor X = Cl, F, OH. De klassificeres som følger18: fluorapatite (FA) Ca10(PO4)6F2, chlorapatite (CA) Ca10(PO4)6Cl2 og hydroxyapatit (HA) Ca10(PO4 )6(OH)2.
Valg af osteoblastdannelse cellelinjer til at fremkalde mineral dannelse er afgørende, da hver cellelinje udstiller en særskilt profil af mineralisering. I denne betænkning, vi sammenlignede Nukleering af mineraler af to valgte menneskelige cell modeller af mineralisering: osteoblastic hFOB 1,19 celler og osteosarkom Saos-2 celler. Osteosarkom-afledte celler er almindeligt anvendt som osteoblastic modeller og Saos-2 celler har bevaret de mest modne osteoblastic karakter19 , mens udifferentierede menneskelige føtal hFOB celler er almindeligt anvendt som en model for normal osteoblastic differentiering20. Deres mineralisering profiler blev analyseret ved hjælp af forskellige metoder: Alizarin rød-S (AR-S) farvning, ultraviolet (UV) lys visualisering, transmissions Elektron Mikroskopi (TEM) imaging, energy dispersive X-ray mikroanalyser (EDX) kvantitering og ion kortlægning. Fordel af TEM-EDX over alternative teknikker, der anvendes i tidligere undersøgelser er, at det giver kvantitative og kvalitative resultater af ion udskiftning i apatit krystaller4,5,21. Det overordnede mål med hjælp af TEM-EDX var at finde en enkel metode til billedbehandling og kvantificering af distribution af Ca, F og Cl-ioner i forskellige mineraler fra forskellige typer af celler i forskellige faser af mineralisering proces. Denne metode har held været anvendt, for eksempel til overvågning af samspillet mellem zink nanopartikler med sameksisterende kemikalier og deres kombinerede virkninger på akvatiske organismer22. I en anden undersøgelse, en kobber photocatalyst på titanium materialer i vandige løsning var udstrakt grad karakteriseret ved hjælp af Induktivt koblet plasma optical emission massespektrometri (ICP-OES), N2 physisorption (BET), XRD, UV-vis DRS, FT-IR, Raman spektroskopi, TEM-EDX og photoelectrochemical målinger23. Vores mål var at sammenligne oprindelse og egenskaber af vesikler og mineraler i to cellelinjer til at forstå den mekanisme, der styrer mineralisering under ossøse differentiering.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57423/57423fig1.jpg"> Figur 1 . Ordningen af de indledende trin i mineralisering i ossøse celler der involverer syntesen af ekstracellulære matrix (ECM) proteiner og frigivelse af matrix vesikler (MVs) fra membranen. MVs ophobes calcium gennem handling af calcium-bindende proteiner, annexins og fosfat, gennem handling af en uorganisk fosfat transporter (PiT) efterfulgt af aktiviteten af væv uspecifikke alkalisk fosfatase (TNAP), som dephosphorylates PPjeg til Pjeg, dermed at lette apatit Nukleering. Derefter, MVs smuldre og frigive apatit til den ekstracellulære medium. Mineralisering er påført den konstant tilførsel af P,jeg og Ca2 + i den ekstracellulære medium4,5. <a href="https://www.jove.com/files/ftp_upload/57423/57423fig1large.jpg" target="_blank">Venligst klik her for at se en større version af dette tal.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#8217;s DMEM/F12 media mixture</td>
			<td>PAA</td>
			<td>E15-813</td>
			<td>1:1, for human fetus hFOB 1.19 SV40 large T antigen transfected osteoblasts (ATCC CRL-11372)</td>
		</tr>
		<tr>
			<td>McCoy&#8217;s 5A medium</td>
			<td>PAA</td>
			<td>E82312-0025</td>
			<td>for human osteosarcoma Saos-2 cells (ATCC HTB-85)</td>
		</tr>
		<tr>
			<td>Antibiotics mixture (penicillin/streptomycin)</td>
			<td>Sigma</td>
			<td>P0781-100ML</td>
			<td>100 U/mL each</td>
		</tr>
		<tr>
			<td>G-418</td>
			<td>Sigma</td>
			<td>68168</td>
			<td>0.3 mg/mL</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10270</td>
			<td>10% for hFOB 1.19 and 15% for Saos-2</td>
		</tr>
		<tr>
			<td>AA</td>
			<td>Sigma</td>
			<td>A-5960</td>
			<td>50 &#181;g/mL</td>
		</tr>
		<tr>
			<td>&#223;-GP</td>
			<td>Sigma</td>
			<td>G9422-100G</td>
			<td>7.5 mM</td>
		</tr>
		<tr>
			<td>Bio-Gel HTP Gel</td>
			<td>Bio-Rad</td>
			<td>130-0420</td>
			<td>for HA</td>
		</tr>
		<tr>
			<td>FA</td>
			<td></td>
			<td></td>
			<td>synthesized by us</td>
		</tr>
		<tr>
			<td>CA</td>
			<td></td>
			<td></td>
			<td>synthesized by us</td>
		</tr>
		<tr>
			<td>Sodium phosphate buffer Na2HPO4/NaH2PO4 mixture</td>
			<td>Sigma</td>
			<td>S7907/S8282</td>
			<td>0.1 M, pH 7.2</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td>pH 7.0, prepared by us</td>
		</tr>
		<tr>
			<td>AR-S in PBS</td>
			<td>Sigma</td>
			<td>A5533-25G</td>
			<td>0.5 g/100 mL, pH 5.0</td>
		</tr>
		<tr>
			<td>Collagenase type IA</td>
			<td>Sigma</td>
			<td>C2674</td>
			<td>500 U/mL</td>
		</tr>
		<tr>
			<td>SCL buffer</td>
			<td></td>
			<td></td>
			<td>prepared by us</td>
		</tr>
		<tr>
			<td>Deionized wather</td>
			<td></td>
			<td></td>
			<td>produced by us</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>POCh</td>
			<td>BA6480111</td>
			<td>absolut 99.8% and solutions 25, 50, 75, 90%</td>
		</tr>
		<tr>
			<td>Uranyl acetate in 50% ethanol</td>
			<td>Polysciences Inc.</td>
			<td>21447-25</td>
			<td>0.25 g/10 mL</td>
		</tr>
		<tr>
			<td>PD medium</td>
			<td></td>
			<td></td>
			<td>pH 7.4, prepared by us</td>
		</tr>
		<tr>
			<td>Fixation mixture (paraformaldehyde/glutaraldehyde)</td>
			<td>Sigma</td>
			<td>158127/G-6257</td>
			<td>3%:1%</td>
		</tr>
		<tr>
			<td>Post-fixation OsO4</td>
			<td>Sigma</td>
			<td>75633</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>LR White resin in ethanol</td>
			<td>Polysciences Inc.</td>
			<td>17411-MUNC 500g</td>
			<td>1:2, 1:1, 100%</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>CHEMPUR</td>
			<td>111024800</td>
			<td>pure</td>
		</tr>
		<tr>
			<td>Tool</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic vials</td>
			<td>Corning Inc.</td>
			<td>430487</td>
			<td>1.2 mL</td>
		</tr>
		<tr>
			<td>Plastic Petri culture dishes</td>
			<td>Falcon</td>
			<td>353003</td>
			<td>100 mm</td>
		</tr>
		<tr>
			<td>Plastic tubes</td>
			<td>Falcon</td>
			<td>352096 and 352070</td>
			<td>15 and 50 mL</td>
		</tr>
		<tr>
			<td>Serological pipettes</td>
			<td>Falcon and VWR</td>
			<td>357521 and 612-3700</td>
			<td>1 and 10 mL</td>
		</tr>
		<tr>
			<td>Plastic microcentrifuge tubes</td>
			<td>Sigma</td>
			<td>Z688312 and Z628034</td>
			<td>1.5 mL black and 2 mL transparent</td>
		</tr>
		<tr>
			<td>Plastic tips</td>
			<td>VWR</td>
			<td>613-0364, 613-0239 and 613-1050</td>
			<td>0.1-10 &#181;L natural, 1-200 &#181;L yellow and 200-1000 &#181;L blue</td>
		</tr>
		<tr>
			<td>Plastic racks</td>
			<td>Light Labs</td>
			<td>A-7055-Z, A-7053-C</td>
			<td>green for tubes, orange for micro tubes and blue for TEM probes</td>
		</tr>
		<tr>
			<td>Laminar Hera Save</td>
			<td>Thermo Scientific Co.</td>
			<td>KS12</td>
			<td>HEPA filter (H14 according to DIN EN 1822)</td>
		</tr>
		<tr>
			<td>Incubators Hera Cell</td>
			<td>Thermo Scientific Co.</td>
			<td>150</td>
			<td>34&#176;C for hFOB 1.19 and 37&#176;C for Saos-2</td>
		</tr>
		<tr>
			<td>Fume hood</td>
			<td>POLON</td>
			<td>WCS-2</td>
			<td>for TEM stainings</td>
		</tr>
		<tr>
			<td>Glass bottles</td>
			<td>SIMAX</td>
			<td>1632414501050 and 1632414501100</td>
			<td>50 and 100 mL</td>
		</tr>
		<tr>
			<td>Quartz glass tubes</td>
			<td>SIMAX</td>
			<td>638422010100</td>
			<td>&#216; 10 mm, L 100 mm</td>
		</tr>
		<tr>
			<td>Pump</td>
			<td>IBS Integra Biosciences</td>
			<td>VACUSAFE comfort</td>
			<td>for vacuum</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Memmert</td>
			<td>UNE 400</td>
			<td>56&#176;C</td>
		</tr>
		<tr>
			<td>Porcelain multi-well plate</td>
			<td>Rosenthal technik</td>
			<td>229/12</td>
			<td>12 wells</td>
		</tr>
		<tr>
			<td>Glass beakers</td>
			<td>SIMAX</td>
			<td>632417010025</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>Glass bottles</td>
			<td>Pocord</td>
			<td>DIN22</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Plastic box</td>
			<td>Agar Scientific Ltd.</td>
			<td></td>
			<td>for darkness</td>
		</tr>
		<tr>
			<td>Snap Fit Gelatin Capsules</td>
			<td>Agar Scientific Ltd.</td>
			<td>G3741</td>
			<td>size 1</td>
		</tr>
		<tr>
			<td>Formvar/Carbon 300 Mesh Ni grids in box</td>
			<td>Agar Scientific Ltd.</td>
			<td>S162N3</td>
			<td>film on the shiny side</td>
		</tr>
		<tr>
			<td>Silicon cell scraper</td>
			<td>Sigma</td>
			<td>SIAL0010-100EA</td>
			<td>size 1.8/25 cm</td>
		</tr>
		<tr>
			<td>Syringe with needle</td>
			<td>BogMark</td>
			<td>007</td>
			<td>syringe 1 mL 40 U, needle 0.5 x 16</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Chirana</td>
			<td>CH005L</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>MPW Medical Instruments</td>
			<td>MPW-350R</td>
			<td>130 x g and 500 x g</td>
		</tr>
		<tr>
			<td>UV transluminator</td>
			<td>UVP</td>
			<td>M-20</td>
			<td>for visible and UV light</td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>LKB</td>
			<td>NOVA</td>
			<td>700&#197; sections</td>
		</tr>
		<tr>
			<td>Block holder</td>
			<td>LKB</td>
			<td>E6711</td>
			<td>round shape</td>
		</tr>
		<tr>
			<td>Diamond knife</td>
			<td>DiATOME</td>
			<td>Ultra 45&#176;</td>
			<td>size 3</td>
		</tr>
		<tr>
			<td>Eyelash holder</td>
			<td></td>
			<td></td>
			<td>bovine, prepared by us</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>ROTH</td>
			<td>2855.1</td>
			<td>antistatic for grids</td>
		</tr>
		<tr>
			<td>Spatulas set</td>
			<td>ROTH</td>
			<td>E286.1</td>
			<td>antistatic for powders</td>
		</tr>
		<tr>
			<td>Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Light Microscope</td>
			<td>Zeiss with Canon</td>
			<td>AxioObserver Z1 equipped with PowerShot G9</td>
			<td>Phase contrast, Transmitted light, 20 x objective, RGB filters</td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>TEM Jeol Co. with Oxford Instruments and SiS-Olympus</td>
			<td>JEM-1400 TEM equipped with full range INCA Energy Dispersive X-ray microanalysis (EDX) System and 11 Megapixel MORADA G2 camera</td>
			<td>magnification 50,000X for TEM and 15,000X for STEM and EDX</td>
		</tr>
		<tr>
			<td>Camera body and lenses</td>
			<td>Nikon</td>
			<td>Nikon D7100 
			Nikkor AF Micro 105 mm f/2.8D 
			Nikkor AF-S 50 mm f/1.8G 
			Nikkor AF 28 mm f/2.8D
</td>
			<td>for movie recordings</td>
		</tr>
		<tr>
			<td>Microphone</td>
			<td>MXL Mics</td>
			<td>Tempo</td>
			<td>for voice recordings</td>
		</tr>
	</tbody>
</table>

analysis of minerals produced by hfob 1.19 and saos-2 cells using transmission electron microscopy with energy dispersive x-ray microanalysis

Denne video præsenterer brugen af transmissions Elektron Mikroskopi med energy dispersive X-ray mikroanalyser (TEM-EDX) til at sammenligne tilstanden af mineraler i vesikler udgivet af to menneskelige knogler cellelinjer: hFOB 1,19 og Saos-2. Disse cellelinjer, efter behandling med ascorbinsyre (AA) og β-glycerophosphate (β-GP), underkastes komplet osteogenic transdifferentiation fra spredning til mineralisering og producere matrix vesikler (MVs) der udløser apatit Nukleering i den ekstracellulær matrix (ECM).
Baseret på Alizarin rød-S (AR-S) farvning og analyse af sammensætningen af mineraler i cellelysater ved hjælp af ultraviolet (UV) lys eller blærer ved hjælp af TEM imaging efterfulgt af EDX kvantitering og ion kortlægning, kan vi udlede at osteosarkom Saos-2 og osteoblastic hFOB 1,19 celler afslører særskilte mineralisering profiler. SAOS-2 celler mineralize mere effektivt end hFOB 1,19 celler og producerer større mineralforekomster, der ikke er synlige i UV-lys, men ligner hydroxyapatit (HA) i, at de har mere Ca og F erstatninger.
Resultaterne ved hjælp af disse teknikker tillader os at konkludere, at processen med mineralisering varierer afhængigt af hvilken celle. Vi foreslår, at på celleniveau, oprindelse og egenskaber af vesikler forudbestemme typen af mineraler.

TEM-EDX giver mulighed for in vitro- imaging af matrix vesikler (MVs) udgivet af mineralizing celler og mineraler produceret af Mvs resultaterne ved hjælp af denne teknik viser, at processen med mineralisering kan fortsætte anderledes i forskellige typer af celler. To cellelinier modtaget den samme osteoblastic transdifferentiation behandling, men stimuleret Saos-2 celler mineraliseret mere effektivt end hFOB 1,19 osteoblaster, som det fremgår af AR-S farvning (<stron...

Watch this Scientific Journal Video about Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis at JoVE.com

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

Vi præsenterer en protokol for at sammenligne tilstanden af mineraler i vesikler udgivet af to menneskelige knogler cellelinjer: hFOB 1,19 og Saos-2. Deres mineralisering profiler blev analyseret af Alizarin rød-S (AR-S) farvning, ultraviolet (UV) lys visualisering, transmissions Elektron Mikroskopi (TEM) billeddannelse og energy dispersive X-ray mikroanalyser (EDX).

Analyse af mineraler produceret af hFOB 1,19 og Saos-2 celler ved hjælp af transmissions elektronmikroskopi med Energy Dispersive X-ray mikroanalyser

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in ...

Research

JoVE Journal

Chemistry

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Kvantificere X-Ray fluorescens Data ved hjælp af kort

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Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

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