For transcript analysis, fresh bone samples were crushed and digested with Collagenase to prepare a single-cell suspension. Pure ECs were sorted by flow cytometry using a FACSAria (BD Biosciences) directly into the lysis buffer of RNeasy Mini Kit (Qiagen). Total RNA was isolated following the manufacturer's protocol and, subsequently, a total of 100–500 ng RNA was used to generate cDNA with the iScript cDNA Synthesis System (Bio-Rad). Further, quantitative PCR (qPCR) was performed using TaqMan gene expression assays on an ABI PRISM 7900HT Sequence Detection System. TaqMan Gene Expression Master Mix (Applied Biosystems) was used to perform qPCR for the FAM-conjugated TaqMan probes Pecam1, Nos3, Dll4, Jag1, Hes1, Hey1, Hey2, Hes5, Angpt2, Cdh5, Sp7, Bglap, Tgfb1, Tgfb2, Tgfb3, Bmp2, Bmp4, Fgf1, Pdgfb and Wnt10b commercially available at Life Technologies (Thermo Fisher Scientific). All the gene expression assays were normalized using endogenous VIC-conjugated Actb probes. For the analysis of Bgalp and Sp7 expression, RNA was isolated from whole-bone samples. Freshly dissected bones were immediately flushed (to remove haematopoietic cells), crushed and lysed in lysis buffer for 15 min before proceeding for RNA isolation, cDNA preparation and qPCR analysis as described above.
Bglap
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Bglap is a piece of laboratory equipment designed to measure the levels of the protein Osteocalcin (also known as bone gamma-carboxyglutamic acid-containing protein or BGLAP) in biological samples. Osteocalcin is a protein secreted by osteoblasts and is commonly used as a biomarker for bone formation. The Bglap instrument provides a quantitative analysis of Osteocalcin concentrations in samples.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Runx2, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Iscript cdna synthesis system, by Bio-Rad (1 mentions)
Tgfb1, by Thermo Fisher Scientific (1 mentions)
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (1 mentions)
Pdgf b, by Thermo Fisher Scientific (1 mentions)
Tgfb2, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
Facsaria, by BD (1 mentions)
Col1 α1, by Thermo Fisher Scientific (1 mentions)
Taqman fast advance master mix, by Thermo Fisher Scientific (1 mentions)
Taqman assay, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Col1a1, by Thermo Fisher Scientific (1 mentions)
Taqman probe set, by Thermo Fisher Scientific (1 mentions)
Quantifast probe pcr kit, by Qiagen (1 mentions)
Superscript 2 reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Mastercycler realplex2, by Eppendorf (1 mentions)
5 protocols using bglap
1
Endothelial Cell Transcript Analysis
2
Quantitative Real-Time PCR for Bone Biology
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The vertebral body of the L3 and L6 (trabecular bone) and the flushed mid-diaphyseal tibias (cortical bone) were stored at −80°C in RNAlater post killing. RNA was extracted using TRIzol reagent (Life Technologies) followed by the RNeasy mini kit (Qiagen). Single-strand cDNA was synthesized using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Quantitative real-time PCR (qPCR) analyses were performed by using predesigned Taqman Assays and Taqman Fast Advance Master Mix (Applied Biosystems). The following predesigned real-time PCR assays were used for gene expression analysis: Acp5 (Trap; Mm00475698_m1), Ctsk (Mm00484036_m1), Tnfsf11 (Rankl; Mm00441908_m1), Alpl (Alp; Mm00475834_m1), Tnfrs11a (Rank; Mm00437135_m1), Tnfrsf11b (Opg; Mm0043545_m1), Bglap (Osteocalcin; Mm01741771_g1), Col1α1 (Mm00801666_g1), Runx2 (Mm00501580_m1), Sp7 (Osterix; Mm04209856_m1) (Applied Biosystems). The house-keeping gene 18S was used as the endogenous control in all analyses. Data are displayed as fold change relative to control.
3
Quantitative PCR Analysis of Osteoblast Differentiation
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One µg of total RNA was reverse-transcribed with Superscript II Reverse Transcription Kit (Invitrogen) and qtPCR was performed using primer and TaqMan probe sets (Applied Biosystems, Foster City, CA) and QuantiFast Probe PCR kit (Qiagen, Valencia, CA) on a Mastercycler realplex2 (Eppendorf, Westbury, NY). Amplification conditions were 95°C for 3 min, followed by 40 cycles at 95°C for 3 s and 60°C for 30 s. Taqman Primer and probe sets AnxA2, AnxA5, Col1a1, Ibsp, Runx2, Sp7, Bglap, and TubA were purchased from Applied Biosystems. Quantitative PCR results were normalized to TubA transcript level for MC3T3-E1 cells to Si controls to obtain 2−ΔΔCt[45] (link).
4
Osteoblast Differentiation Transcriptional Profile
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Total RNA was extracted from MC3T3-E1 cells cultured in osteoblast differentiation medium for 12 days using the same method as described above and mouse maxillary palatal gingiva using the TRI reagent (Molecular Research Center, Inc., Cincinnati, OH, USA); RNA was quantified via spectrophotometry at 260 and 280 nm. RNA was reverse-transcribed using SuperScript VILO Ⅳ Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). qPCR was performed using the cDNA in a StepOnePlus real-time PCR system (Thermo Fisher Scientific) according to the manufacturer’s protocol. The data were analyzed using the comparative CT (ΔΔCt) method. TaqMan probes, sense primers, and antisense primers for the expression of a housekeeping gene (glyceraldehyde 3-phosphate dehydrogenase, assay ID: 4331182 Mm99999915_g1), along with Runx2 (assay ID: 4331182 Mm00501584_m1), Sp7 (osterix, assay ID: 4331182 Mm00504574_m1), bone γ-carboxyglutamic acid (Bglap, assay ID: 4331182 Mm00649782_gH), epidermal growth factor-like repeats and discoidin domains 3 (Edil3; Del1, encoded DEL-1, assay ID: 4331182 Mm01291247_m1), nuclear factor of activated T cells 1 (Nfatc1, assay ID: 4331182 Mm01265944_m1), and tumor necrosis factor receptor superfamily member 11A (Tnfrsf11a; Rank, encoded RANK, assay ID: 4331182 Mm00437132_m1) were purchased from Thermo Fisher Scientific.
5
Immunofluorescent Characterization of Cell Sheets
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Engineered cell sheets were fixed in 10% neutral buffered formalin and embedded in paraffin. Tissue sections were then stained with hematoxylin & eosin (H&E) or used for immunofluorescent staining with CD19, CD73, Acetyl-CoA1 (NovusBIo, CO, USA), CD29, COLII (Collagen II), HLA-A, HLA-DR (Abcam, MA, USA), CD45R, CD105, BGLAP (Osteocalcin), SREBP1 (ThermoFisher Scientific, CA, USA), PPARg (Cell Signaling, MA, USA), ACAN (Aggrecan) and SPARC (ThermoFisher Scientific). Alexa Fluor R 488 donkey antimouse conjugated second antibodies, Alexa Fluor 488 donkey antirabbit conjugated second antibodies and Alexa Fluor 488 donkey antirat conjugated second antibodies (Invitrogen, OR, USA) were used. Propidium iodine (Invitrogen) was used to stain nuclear DNA. A Nikon 400 fluorescence microscope was used to analyze the slides (Nikon Inc., NY, USA).
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