{"id":23,"date":"2016-09-01T09:37:11","date_gmt":"2016-09-01T16:37:11","guid":{"rendered":"https:\/\/sites.ewu.edu\/jashley6\/?page_id=23"},"modified":"2024-07-16T10:58:31","modified_gmt":"2024-07-16T17:58:31","slug":"preoc-diff","status":"publish","type":"page","link":"https:\/\/test-inside.ewu.edu\/jashley6\/preoc-diff\/","title":{"rendered":"Osteoclast Differentiation"},"content":{"rendered":"<h1 style=\"text-align: center;\"><strong><span style=\"text-decoration: underline;\">Osteoclast precursor culture and differentiation<\/span><\/strong><\/h1>\n<h3><span style=\"text-decoration: underline;\"><strong>Materials<br \/>\n<\/strong><\/span><strong><em>Dissection<\/em><\/strong><\/h3>\n<ul>\n<li>Clean scissors<\/li>\n<li>Clean forceps<\/li>\n<li>70% Ethanol<\/li>\n<li>Dissecting board (cork, Styrofoam, etc.)<\/li>\n<li>Push pins<\/li>\n<li>Absorptive disposable cloth (like bench liner)<\/li>\n<li>10mL syringe (Fisher Scientific &#8211; <a href=\"http:\/\/www.fishersci.com\/shop\/products\/bd-luer-lok-disposable-syringes-without-needles-11\/148232a\">14-823-2A<\/a>)<\/li>\n<li>25-guage needle (Fisher Scientific &#8211; <a href=\"http:\/\/www.fishersci.com\/shop\/products\/bd-precisionglide-single-use-needles-regular-bevel-regular-wall-10\/1482113d\">14-821-13D<\/a>)<\/li>\n<\/ul>\n<h3><em><strong>Culture<br \/>\n<\/strong><\/em><\/h3>\n<ul>\n<li>\u03b1-MEM\n<ul>\n<li>Stericup 0.22-micron PVDF filter with 1L bottle (Millipore &#8211;\u00a0<a href=\"https:\/\/www.emdmillipore.com\/US\/en\/product\/Stericup-GV%2C-0.22%C2%A0%C2%B5m%2C-PVDF%2C-10001000%C2%A0mL%2C-radio-sterilized,MM_NF-SCGVU11RE;pgid=WZtMKfaYs81SRpEoh9VgbI720000itMOfC4H;sid=oNJlovW3Sot5oqJ_wM91KhviGVSn_Qgo5gJXVauXSqWDun3ZeBB4pUUxpWNraJa8TgpLuybBY--cCkj3eqTZ-wEBwnPhqzVCsJorfekxh8nHpv_Mewv8kK4hr9iyXPrxlQ7tzC11KLy9YDaKWXrtzC11\">SCGVU11RE<\/a>)<\/li>\n<li>1 jar Minimum Essential Medium powder (Sigma-Aldrich &#8211; \u00a0<a href=\"http:\/\/www.sigmaaldrich.com\/catalog\/product\/sigma\/m0894\">M0894-10X1L<\/a>)<\/li>\n<li>880mL deionized water<\/li>\n<li>10mL 200mM L-glutamine (ThermoFisher &#8211;\u00a0<a href=\"https:\/\/www.thermofisher.com\/order\/catalog\/product\/25030081\">25030081<\/a>).<\/li>\n<li>10mL 10,000IU Penicillin\/10,000\u03bcg Streptomycin (ThermoFisher &#8211; <a href=\"https:\/\/www.thermofisher.com\/order\/catalog\/product\/15140122\"><span class=\"ng-binding\">15140122<\/span><\/a>)<\/li>\n<li>1.98g Sodium Bicarbonate (Fisher Scientific &#8211; <a href=\"https:\/\/www.fishersci.com\/shop\/products\/sodium-bicarbonate-fine-white-powder-fisher-bioreagents-2\/bp328500\">BP328-500<\/a>)<\/li>\n<li>Add 100mL <a href=\"http:\/\/www.gembio.com\/post\/heat-inactivation-serum\">heat inactivated<\/a> fetal bovine serum<\/li>\n<li>Mix and sterile filter into a new bottle<\/li>\n<\/ul>\n<\/li>\n<li>Freezing medium\n<ul>\n<li>90%\u00a0\u03b1-MEM<\/li>\n<li>10% Dimethyl sulfoxide (Fisher Scientific &#8211; <a href=\"http:\/\/www.fishersci.com\/shop\/products\/dimethyl-sulfoxide-fisher-bioreagents-3\/bp231100\">BP231-100<\/a>)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<ul>\n<li>Phosphate Buffered Saline without Ca<sup>++<\/sup> and Mg<sup>++<\/sup> (ThermoFisher &#8211; <a href=\"http:\/\/www.fishersci.com\/shop\/products\/gibco-dpbs-without-calcium-magnesium-7\/14190235\"><span class=\"ng-binding\">14190235<\/span><\/a>)<\/li>\n<li>100mm tissue culture-treated dishes (Denville Scientific &#8211; <a href=\"https:\/\/www.denvillescientific.com\/products\/denville%C2%AEcell-culture-dishes--100mm-cell-culture-dish--thickness-20mm-qty--bag-5-qty--case-300\">T1110<\/a>)<\/li>\n<li>60mm untreated suspension culture dishes (Fisher Scientific &#8211; <a href=\"http:\/\/www.fishersci.com\/shop\/products\/corning-culture-dishes-4\/0877231\">08-772-31<\/a>)<\/li>\n<li>24-well tissue culture plates (Denville Scientific &#8211;\u00a0<a href=\"https:\/\/www.denvillescientific.com\/products\/denville%C2%AE-cell-culture-plates--24-well-cell-culture-plate--flat-bottom-qty--bag-1-qty--case-50\">T1024<\/a>)<\/li>\n<li>50mL and 15mL tubes<\/li>\n<li>5mL and 10mL serological pipettes<\/li>\n<li>37<sup>o<\/sup>C water bath<\/li>\n<li>ACK lysing buffer (ThermoFisher &#8211;\u00a0<span class=\"ng-binding\"><a href=\"https:\/\/www.thermofisher.com\/order\/catalog\/product\/A1049201\">A1049201<\/a>)<\/span><\/li>\n<li>Recombinant mouse Monocyte\/Macrophage Colony-Stimulating Factor\u00a0(MCSF) (Biolegend &#8211; <a href=\"http:\/\/www.biolegend.com\/recombinant-mouse-m-csf-carrier-free-7780.html\">576406<\/a>)<\/li>\n<li>Recombinant mouse Receptor Activator of Nuclear Factor \u03baB Ligand (RANKL) (Biolegend &#8211; <a href=\"https:\/\/www.biolegend.com\/en-us\/products\/recombinant-mouse-trance-rankl-tnfsf11-carrier-free-15254\">769404<\/a>)<\/li>\n<li>Accutase cell dissociation reagent (ThermoFisher &#8211;\u00a0<span class=\"ng-binding\"><a href=\"https:\/\/www.thermofisher.com\/order\/catalog\/product\/A1110501\">A1110501<\/a>)<\/span><\/li>\n<li>Trypan blue<\/li>\n<\/ul>\n<h3><span style=\"text-decoration: underline;\"><strong>Method<br \/>\n<\/strong><\/span><em><strong>Day 1<\/strong><\/em><\/h3>\n<ol>\n<li>Sacrifice a mouse (C57Bl\/6 work well) via CO<sub>2<\/sub> inhalation followed by cervical dislocation<\/li>\n<li>Using clean technique, dissect out and separate the tibiae and femora.<\/li>\n<li>Cut the ends off femora and tibiae to facilitate flushing<\/li>\n<li>Working in the hood, fill a 10mL syringe with 10mL \u03b1-MEM.<\/li>\n<li>Pick up a bone with forceps and insert syringe needle into one end.<\/li>\n<li>Flush ~2.5mL \u03b1-MEM through the bone into a 15mL tube. Note: you\u2019ll know you\u2019ve flushed out the marrow if the bone changes from a reddish color to a more white color.<\/li>\n<li>Repeat the flushing procedure for remaining bones adding all marrow flushes from each mouse into a single tube.<\/li>\n<li>Using a 10mL serological pipette, transfer medium with marrow cells into a clean 15mL tube.\u00a0Take care not to transfer any debris from the bone that may have settled to the bottom of the tube.<\/li>\n<li>Pellet cells at 300RCF for 5 minutes.<\/li>\n<li>Aspirate medium and resuspend pellet in 500\u03bcL ACK lysing buffer and incubate at 37<sup>o<\/sup>C for 2 minutes to lyse red blood cells.<\/li>\n<li>Add 10mL PBS to cells and pellet at 300RCF for 5 minutes.<\/li>\n<li>Resuspend pellet in 10mL\u00a0\u03b1-MEM and transfer to a 100mm tissue culture-treated dish.<\/li>\n<li>Incubate plate in a 37<sup>o<\/sup>C humidified incubator at 5% CO<sub>2<\/sub> This will allow osteoblasts, stroma, fibroblasts, and all other adherent cells that may be in the flush to attach to the plate.<\/li>\n<\/ol>\n<h3><em><strong>Day 2<\/strong><\/em><\/h3>\n<ol>\n<li>Transfer non-adherent cells to a 15mL tube.<\/li>\n<li>Add \u03b1-MEM to a volume of 10mL.<\/li>\n<li>Add MCSF to a final concentration of 25ng\/mL.<\/li>\n<li>Deposit cells to 100mm petri dish (non-culture treated, non-pyrogenic).<\/li>\n<li>Incubate dishes\u00a0in a 37<sup>o<\/sup>C humidified incubator at 5% CO<sub>2<\/sub> overnight.<\/li>\n<\/ol>\n<h3><em><strong>Day 3<\/strong><\/em><\/h3>\n<ol>\n<li>Refresh medium on cells.<\/li>\n<\/ol>\n<h3><em><strong>Day 5<\/strong><\/em><\/h3>\n<ol>\n<li>When plates are 75% confluent, wash with PBS, and add 1mL Accutase. It usually takes ~5min at room temperature for Accutase to weaken the attachment enough to lift the cells.<\/li>\n<li>Add 4mL \u03b1-MEM to cells and use gentle pipette motion to lift. Move cells to a 15mL tube.<\/li>\n<li>Make a 2-fold dilution of cells (15\u03bcL cells + 15\u03bcL trypan blue) and count cells using a hemocytometer.<\/li>\n<li>To make osteoclasts in the following plates use these numbers of macrophages (~26000 cells per cm<sup>2<\/sup> of culture area):\n<ul>\n<li>96-well culture treated plate \u2013 10 x 10<sup>3 <\/sup>(seed in 200\u03bcL)<\/li>\n<li>24-well culture treated plate \u2013 5 x 10<sup>4 <\/sup>(seed in 1mL)<\/li>\n<li>35mm culture treated dish \u2013 2.5 x 10<sup>5<\/sup> (seed in 2mL)<\/li>\n<li>60mm culture treated dish \u2013 7.4 x 10<sup>5 <\/sup>(seed in 3mL)<\/li>\n<li>100mm culture treated dish \u2013 2 x 10<sup>6<\/sup> (seed in 10mL)<\/li>\n<\/ul>\n<\/li>\n<li>Prepare a sufficient volume of cells for seeding and add MCSF to a final concentration of 25ng\/mL and RANKL to a concentration between 6.25 and 25ng\/mL.\n<ul>\n<li>In our experience, 25ng\/mL RANKL produces maximal osteoclast differentiation. If you expect your experimental group to demonstrate enhanced differentiation, use a lower concentration.<\/li>\n<\/ul>\n<\/li>\n<li>Alternatively, cells can be seeded on bone or dentine slices. Use the same cell density of the culture vessel the slice is kept in (usually a 24-well plate)<\/li>\n<li>Incubate dishes\u00a0in a 37<sup>o<\/sup>C humidified incubator at 5% CO<sub>2<\/sub>\u00a0for 2 days.<\/li>\n<\/ol>\n<h3><em><strong>Day 6<\/strong><\/em><\/h3>\n<ol>\n<li>Refresh the medium on the cells &#8211; \u03b1-MEM w\/ 25ng\/mL M-CSF + 6.25-25ng\/mL RANKL<\/li>\n<li>Late in the afternoon, you may see some multinuclear cells.<\/li>\n<\/ol>\n<h3><em><strong>Day 7<\/strong><\/em><\/h3>\n<ol>\n<li>Osteoclastogenesis should be complete in the AM<\/li>\n<li>Osteoclasts don\u2019t live long in culture, so watch your cells closely.<\/li>\n<li>Osteoclasts on bone slices usually take 1 more day to mature than those on culture plastic.<\/li>\n<li>Osteoclasts can be identified using a TRAP stain (<a href=\"https:\/\/www.urmc.rochester.edu\/MediaLibraries\/URMCMedia\/musculoskeletal-research\/core-services\/histology\/documents\/CMSR-TRAP-stain.pdf\">sample staining protocol<\/a>).<\/li>\n<\/ol>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"size-medium wp-image-29 aligncenter\" src=\"https:\/\/test-inside.ewu.edu\/jashley6\/wp-content\/uploads\/sites\/226\/2016\/09\/Bl6-100-300x300.jpg\" alt=\"Bl6 - 100\" width=\"300\" height=\"300\" srcset=\"https:\/\/test-inside.ewu.edu\/jashley6\/wp-content\/uploads\/sites\/226\/2016\/09\/Bl6-100-300x300.jpg 300w, https:\/\/test-inside.ewu.edu\/jashley6\/wp-content\/uploads\/sites\/226\/2016\/09\/Bl6-100-150x150.jpg 150w, https:\/\/test-inside.ewu.edu\/jashley6\/wp-content\/uploads\/sites\/226\/2016\/09\/Bl6-100-768x768.jpg 768w, https:\/\/test-inside.ewu.edu\/jashley6\/wp-content\/uploads\/sites\/226\/2016\/09\/Bl6-100-1024x1024.jpg 1024w, https:\/\/test-inside.ewu.edu\/jashley6\/wp-content\/uploads\/sites\/226\/2016\/09\/Bl6-100.jpg 1080w\" sizes=\"(max-width: 300px) 100vw, 300px\" \/><\/p>\n<p style=\"text-align: center;\">Mature osteoclasts stained for Tartrate-Resistant Acid Phosphatase (TRAP) activity.<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Osteoclast precursor culture and differentiation Materials Dissection Clean scissors Clean forceps 70% Ethanol Dissecting board (cork, Styrofoam, etc.) Push pins Absorptive disposable cloth (like bench liner) 10mL syringe (Fisher Scientific &#8211; 14-823-2A) 25-guage needle (Fisher Scientific &#8211; 14-821-13D) Culture \u03b1-MEM Stericup 0.22-micron PVDF filter with 1L bottle (Millipore &#8211;\u00a0SCGVU11RE) 1 jar Minimum Essential Medium powder &#8230; <a title=\"Osteoclast Differentiation\" class=\"read-more\" href=\"https:\/\/test-inside.ewu.edu\/jashley6\/preoc-diff\/\" aria-label=\"Read more about Osteoclast Differentiation\">Read more<\/a><\/p>\n","protected":false},"author":66,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"wpo365_audiences":[],"wpo365_private":false,"footnotes":""},"class_list":["post-23","page","type-page","status-publish"],"_links":{"self":[{"href":"https:\/\/test-inside.ewu.edu\/jashley6\/wp-json\/wp\/v2\/pages\/23"}],"collection":[{"href":"https:\/\/test-inside.ewu.edu\/jashley6\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/test-inside.ewu.edu\/jashley6\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/test-inside.ewu.edu\/jashley6\/wp-json\/wp\/v2\/users\/66"}],"replies":[{"embeddable":true,"href":"https:\/\/test-inside.ewu.edu\/jashley6\/wp-json\/wp\/v2\/comments?post=23"}],"version-history":[{"count":2,"href":"https:\/\/test-inside.ewu.edu\/jashley6\/wp-json\/wp\/v2\/pages\/23\/revisions"}],"predecessor-version":[{"id":97,"href":"https:\/\/test-inside.ewu.edu\/jashley6\/wp-json\/wp\/v2\/pages\/23\/revisions\/97"}],"wp:attachment":[{"href":"https:\/\/test-inside.ewu.edu\/jashley6\/wp-json\/wp\/v2\/media?parent=23"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}