collagenase type i  (Thermo Fisher)


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    Name:
    Collagenase Type I powder
    Description:
    Collagenase is a protease that cleaves the bond between a neutral amino acid X and glycine in the sequence Pro X Gly Pro which is found with high frequency in collagen Collagenase is unique among proteases in its ability to degrade the triple helical native collagen fibrils commonly found in connective tissues such as skin tendon blood vessels and bone Collagenase disaggregation is suitable for the culture of human tumors mouse kidney human adult and fetal brain and many other tissues including epithelium Collagenase is relatively gentle dissociates well at physiological temperature and pH and requires no mechanical agitation or special equipment Gibco Collagenase Type I is isolated from Clostridium histolyticum and packaged as a lyophilized non sterile powder for research use in cell or tissue dissociation and organ perfusions Gibco Collagenase Type I activity is guaranteed to be greater than 125 units mg Compared to other collagenase preparations Gibco Collagenase Type I has average levels of collagenase caseinase clostripain and tryptic activities and is well suited for the digestion of fat adrenal and liver cells or tissues
    Catalog Number:
    17018029
    Price:
    None
    Category:
    Cell Culture Transfection Reagents
    Applications:
    Cell Culture|Mammalian Cell Culture
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    Structured Review

    Thermo Fisher collagenase type i
    Collagenase is a protease that cleaves the bond between a neutral amino acid X and glycine in the sequence Pro X Gly Pro which is found with high frequency in collagen Collagenase is unique among proteases in its ability to degrade the triple helical native collagen fibrils commonly found in connective tissues such as skin tendon blood vessels and bone Collagenase disaggregation is suitable for the culture of human tumors mouse kidney human adult and fetal brain and many other tissues including epithelium Collagenase is relatively gentle dissociates well at physiological temperature and pH and requires no mechanical agitation or special equipment Gibco Collagenase Type I is isolated from Clostridium histolyticum and packaged as a lyophilized non sterile powder for research use in cell or tissue dissociation and organ perfusions Gibco Collagenase Type I activity is guaranteed to be greater than 125 units mg Compared to other collagenase preparations Gibco Collagenase Type I has average levels of collagenase caseinase clostripain and tryptic activities and is well suited for the digestion of fat adrenal and liver cells or tissues
    https://www.bioz.com/result/collagenase type i/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Related Articles

    Incubation:

    Article Title: Establishment of primary human breast cancer cell lines using “pulsed hypoxia” method and development of metastatic tumor model in immunodeficient mice
    Article Snippet: Tissues were washed extensively with 1× PBS with 200 U/mL penicillin, 200 μg/mL streptomycin, and 500 mg/mL amphotericin without centrifugation. .. For collagenase treatment tissue specimens were mechanically dissociated using a scalpel with removal of vascular material and transferred to a solution of 20 mg/mL collagenase I (Gibco BRL Co., Invitrogen) in DMEM media and incubated at 37 °C for 15 h on a shaking incubator (Grant Bio, Keison Products, UK). .. Specimens dissociated into single cells were washed with 10× excess of phosphate-buffered saline (PBS) and cell pellet was collected by centrifugation at 300×g for 5 min.

    Article Title: Twist1 in Infiltrating Macrophages Attenuates Kidney Fibrosis via Matrix Metallopeptidase 13–Mediated Matrix Degradation
    Article Snippet: Adherent cells were changed to serum-free medium and preconditioned with 150 IU/ml IFN γ (catalog 4031; Invitrogen) for 6 hours and then stimulated with 100 ng/ml LPS (catalog L2630; Sigma-Aldrich). .. Monocyte Macrophage Isolation from Obstructed Kidneys Kidneys were harvested and minced, followed by incubation with 0.1% collagenase type 1 (catalog 17100-017; Gibco) for 30 minutes at 37°C. ..

    Article Title: Application of bioactive hydrogels combined with dental pulp stem cells for the repair of large gap peripheral nerve injuries
    Article Snippet: 2.8 Degradation analysis The in vitro degradation rate of GelMA-bFGF hydrogels was determined as previously described [ ]. .. Briefly, lyophilized GelMA-bFGF hydrogels (5, 10 and 15% (w/v)) were weighed (W0 ) and incubated in PBS supplemented with 1 ng/mL of collagenase type I (Gibco, USA). ..

    Article Title: Promotion of Dermal Regeneration using Pullulan/Gelatin Porous Skin Substitute
    Article Snippet: .. To assess biodegradation, PG2 scaffolds (8 mm biopsies) were placed into a 60 μg mL−1 (7.5 unit mL−1 ) collagenase I (Thermo Fisher Scientific) PBS solution, pH = 7.4, and incubated at 37 °C with 5% CO2 and 100% humidity ( ). ..

    Article Title: In vitro comparative study of two decellularization protocols in search of an optimal myocardial scaffold for recellularization
    Article Snippet: The total amount of DNA was quantified using spectrophotometry (NanoDrop ND-1000, NanoDrop Technologies) and normalized to the sample tissue weight (average weight: 8.3-24.7 mg). .. Decellularized lyophilized scaffolds (n = 8) were weighed and incubated with 10 ml of a PBS solution containing 0.1% collagenase I (Gibco) for 24 h at 37°C with gentle stirring [ ]. ..

    Isolation:

    Article Title: Twist1 in Infiltrating Macrophages Attenuates Kidney Fibrosis via Matrix Metallopeptidase 13–Mediated Matrix Degradation
    Article Snippet: Adherent cells were changed to serum-free medium and preconditioned with 150 IU/ml IFN γ (catalog 4031; Invitrogen) for 6 hours and then stimulated with 100 ng/ml LPS (catalog L2630; Sigma-Aldrich). .. Monocyte Macrophage Isolation from Obstructed Kidneys Kidneys were harvested and minced, followed by incubation with 0.1% collagenase type 1 (catalog 17100-017; Gibco) for 30 minutes at 37°C. ..

    Centrifugation:

    Article Title: Lysophosphatidic acid induces YAP-promoted proliferation of human corneal endothelial cells via PI3K and ROCK pathways
    Article Snippet: Under a dissecting microscope, the trabecular meshwork was cleaned, and Descemet’s membranes containing HCECs were stripped using forceps. .. Following digestion at 37 °C for 24 hours with 0.5 mg/ml collagenase A in wash medium, HCEC aggregates were collected via centrifugation at 495 g for 8 minutes to remove the digestion solution and then cultured in 8-well Lab-Tek II chamber slides (Nalge Nunc International, Rochester, NY) coated with a fibronectin-collagen coating mix (AthenaES, Baltimore, MD) in HCEC growth medium, composed of Opti-MEM supplemented with 10% FBS, 20 ng/ml hEGF, 10 ng/ml FGF-basic, RPMI 1640 vitamin (1×) solution (Sigma-Aldrich), 25 µg/ml gentamicin, and 1.25 µg/ml amphotericin B. .. The human corneal endothelial cell line B4G12 (Creative Bioarray, NY) was cultured in B4G12 medium, composed of HESFM supplemented with 2% FBS and 10 ng/ml bFGF.

    Cell Culture:

    Article Title: Lysophosphatidic acid induces YAP-promoted proliferation of human corneal endothelial cells via PI3K and ROCK pathways
    Article Snippet: Under a dissecting microscope, the trabecular meshwork was cleaned, and Descemet’s membranes containing HCECs were stripped using forceps. .. Following digestion at 37 °C for 24 hours with 0.5 mg/ml collagenase A in wash medium, HCEC aggregates were collected via centrifugation at 495 g for 8 minutes to remove the digestion solution and then cultured in 8-well Lab-Tek II chamber slides (Nalge Nunc International, Rochester, NY) coated with a fibronectin-collagen coating mix (AthenaES, Baltimore, MD) in HCEC growth medium, composed of Opti-MEM supplemented with 10% FBS, 20 ng/ml hEGF, 10 ng/ml FGF-basic, RPMI 1640 vitamin (1×) solution (Sigma-Aldrich), 25 µg/ml gentamicin, and 1.25 µg/ml amphotericin B. .. The human corneal endothelial cell line B4G12 (Creative Bioarray, NY) was cultured in B4G12 medium, composed of HESFM supplemented with 2% FBS and 10 ng/ml bFGF.

    other:

    Article Title: Localized co-delivery of collagenase and trastuzumab by thermosensitive hydrogels for enhanced antitumor efficacy in human breast xenograft
    Article Snippet: Collagenase-I was supplied by Invitrogen (Carlsbad, CA) and trastuzumab (Herceptin) was obtained from Roche (Basel, Switzerland).

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  • 99
    Thermo Fisher gene exp col1a1 rn01463848 m1
    mRNA expression in the culture medium on day 14 ( n = 3 for each group). a Expression of (i) OC, (ii) ALP, and ( b ) expression of (i) Runx2, (ii) <t>Col1a1,</t> (iii) Col4a1. The expression of OC mRNA in SrHAP, SrSiP and SrZnSiP group and the expression of ALP mRNA in the SrZnSiP group were significantly higher than that in non-coated PEEK group. However, there was no significant difference with respect to Runx2, Col1a1 and Col4a1 mRNA expressions. Data are shown as the mean ± SD. Asterisk indicates p
    Gene Exp Col1a1 Rn01463848 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher gene exp col1a1 mm00801666 g1
    Induction of <t>Col1a1</t> and Loxl mRNA expression during fibrosis reach the same level in grafts from I l10 −/− and WT donors in the transplant model of fibrosis. ( A ) qPCR confirmed increase of Mcp1 , Ifnγ , inos , Il18 and Il6 in the small bowel from Il10 −/− donor mice compared to WT (*p
    Gene Exp Col1a1 Mm00801666 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher 3d collagen scaffolds
    Assessing mineralization of 4T1 cells grown in an osteogenic cocktail within <t>3D</t> collagen scaffolds. Representative images are shown at 400× and 40× magnifications and the scale bars represent 200 µm and 2000 µm respectively. (A) Positive hematoxylin and eosin (H E) staining was observed at 400× magnification by day 14 and the intensity of the hematoxylin stain increased by day 28. Some positive alizarin red S staining for calcium (red) was detected by day 14. Complete positive staining with alizarin red S and von Kossa (including toluidine blue counterstain) was observed by day 28. (B) At 40× magnification the previously described patterns of staining are confirmed and minor contraction and disintegration of the scaffolds are observed over time. (C) Assessing cell viability of 4T1 cells grown within the 3D scaffolds using an alamar blue metabolic assay. An increase in cell viability was detected between day 14 and day 28. Each time point represent the mean % reduction in alamar blue +/− SEM, n = 3, students t-test. *P = 0.0284 day 28 vs. day 14. OC (osteogenic cocktail) = with 50 µg/ml ascorbic acid and 10 mM β-glycerophosphate.
    3d Collagen Scaffolds, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    mRNA expression in the culture medium on day 14 ( n = 3 for each group). a Expression of (i) OC, (ii) ALP, and ( b ) expression of (i) Runx2, (ii) Col1a1, (iii) Col4a1. The expression of OC mRNA in SrHAP, SrSiP and SrZnSiP group and the expression of ALP mRNA in the SrZnSiP group were significantly higher than that in non-coated PEEK group. However, there was no significant difference with respect to Runx2, Col1a1 and Col4a1 mRNA expressions. Data are shown as the mean ± SD. Asterisk indicates p

    Journal: BMC Musculoskeletal Disorders

    Article Title: In vitro osteogenesis of rat bone marrow mesenchymal cells on PEEK disks with heat-fixed apatite by CO2 laser bonding

    doi: 10.1186/s12891-020-03716-1

    Figure Lengend Snippet: mRNA expression in the culture medium on day 14 ( n = 3 for each group). a Expression of (i) OC, (ii) ALP, and ( b ) expression of (i) Runx2, (ii) Col1a1, (iii) Col4a1. The expression of OC mRNA in SrHAP, SrSiP and SrZnSiP group and the expression of ALP mRNA in the SrZnSiP group were significantly higher than that in non-coated PEEK group. However, there was no significant difference with respect to Runx2, Col1a1 and Col4a1 mRNA expressions. Data are shown as the mean ± SD. Asterisk indicates p

    Article Snippet: The primers for the target mRNAs were OC (Rn01455285 g1; Thermo Fisher Scientific, Waltham, MA, USA), ALP (Rn00564931 m1), Runx2 (Rn01512298 m1), Col1a1 (Rn01463848 m1), Col4a1 (Rn01482925 m1), and GAPDH (Rn99999916 s1).

    Techniques: Expressing

    Induction of Col1a1 and Loxl mRNA expression during fibrosis reach the same level in grafts from I l10 −/− and WT donors in the transplant model of fibrosis. ( A ) qPCR confirmed increase of Mcp1 , Ifnγ , inos , Il18 and Il6 in the small bowel from Il10 −/− donor mice compared to WT (*p

    Journal: Scientific Reports

    Article Title: Severity of local inflammation does not impact development of fibrosis in mouse models of intestinal fibrosis

    doi: 10.1038/s41598-018-33452-5

    Figure Lengend Snippet: Induction of Col1a1 and Loxl mRNA expression during fibrosis reach the same level in grafts from I l10 −/− and WT donors in the transplant model of fibrosis. ( A ) qPCR confirmed increase of Mcp1 , Ifnγ , inos , Il18 and Il6 in the small bowel from Il10 −/− donor mice compared to WT (*p

    Article Snippet: qPCR was performed using Taqman gene expression assays for Il1β Mm01336189_m1, inos Mm01309893_m1, Mcp1 Mm00441242_m1, Ifnγ Mm00439552_s1, Mmp-9 Mm00442991_m1, Timp-1 Mm00441818_m1, Loxl-2 Mm00804740_m1, Col1a1 Mm00801666_g1 and Gapdh 4352339E.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay

    Activation of primary fibroblasts by HCV and inhibition by antibody to TGF-β1 Activation of fibroblast activation markers are primarily inhibted by TGF-β1 specific antibody in primary human hepatic stellate cells (panels A–C). Upregulated activation markers for primary human HSC (α-SMA/Col1A1/CTGF) with conditioned medium (CM) from HCV infected immortalized human hepatocyte are neutralized in presence of TGF-β specific antibodies. Activation markers were analyzed by qRT-PCR. Values represent from three independent experiments ±SD. Statistical significance was analyzed using the two-tailed Student’s t-test: *P

    Journal: Hepatology (Baltimore, Md.)

    Article Title: HEPATITIS C VIRUS INDUCED TUMOR INITIATING CANCER STEM-LIKE CELLS ACTIVATE STROMAL FIBROBLASTS IN XENOGRAFT TUMOR MODEL

    doi: 10.1002/hep.29346

    Figure Lengend Snippet: Activation of primary fibroblasts by HCV and inhibition by antibody to TGF-β1 Activation of fibroblast activation markers are primarily inhibted by TGF-β1 specific antibody in primary human hepatic stellate cells (panels A–C). Upregulated activation markers for primary human HSC (α-SMA/Col1A1/CTGF) with conditioned medium (CM) from HCV infected immortalized human hepatocyte are neutralized in presence of TGF-β specific antibodies. Activation markers were analyzed by qRT-PCR. Values represent from three independent experiments ±SD. Statistical significance was analyzed using the two-tailed Student’s t-test: *P

    Article Snippet: RNA was quantified by using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized by using random hexamers and a Superscript III reverse transcriptase kit (Invitrogen, CA). qRT-PCR was performed with cDNA using TaqMan gene expression PCR master mix and 6-carboxyfluorescein (FAM)-MGB primers for mouse ACTA2 (assay ID, Mm00725412_s1), COL1A2 (assay ID, Mm00483888_m1), COL1A1 (assay ID, Mm00801666_g1), CTGF (assay ID, Mm01192933_g1), Vimentin (assay ID, Mm01333430_m1), FSP-1 (assay ID, Mm00803372_g1), 18S RNA (assay ID, Mm04277571_s1).

    Techniques: Activation Assay, Inhibition, Infection, Quantitative RT-PCR, Two Tailed Test

    Analyses for TAF activation markers in xenograft tumors after implantation of TISCs into NSG mice RNA from xenograft tumors (T1 and T2) was isolated and analyzed for expression of α-SMA, COL1A2, COL1A1, Vimentin, CTGF, and FSP-1 by qRT-PCR and normalized with 18SRNA ( Panel A) . TISCs grown on petridish (C) and control human liver (C1 and C2) RNAs were used for comparison. Species specific analysis of TAF activation markers in xenograft tumors ( panel B) . RNA from T1 or T2 tumors was analyzed for expression of α-SMA and COL1A2 using mouse and human specific primers. Statistical significance of the results was analyzed using the two-tailed Student’s t-test: *P

    Journal: Hepatology (Baltimore, Md.)

    Article Title: HEPATITIS C VIRUS INDUCED TUMOR INITIATING CANCER STEM-LIKE CELLS ACTIVATE STROMAL FIBROBLASTS IN XENOGRAFT TUMOR MODEL

    doi: 10.1002/hep.29346

    Figure Lengend Snippet: Analyses for TAF activation markers in xenograft tumors after implantation of TISCs into NSG mice RNA from xenograft tumors (T1 and T2) was isolated and analyzed for expression of α-SMA, COL1A2, COL1A1, Vimentin, CTGF, and FSP-1 by qRT-PCR and normalized with 18SRNA ( Panel A) . TISCs grown on petridish (C) and control human liver (C1 and C2) RNAs were used for comparison. Species specific analysis of TAF activation markers in xenograft tumors ( panel B) . RNA from T1 or T2 tumors was analyzed for expression of α-SMA and COL1A2 using mouse and human specific primers. Statistical significance of the results was analyzed using the two-tailed Student’s t-test: *P

    Article Snippet: RNA was quantified by using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized by using random hexamers and a Superscript III reverse transcriptase kit (Invitrogen, CA). qRT-PCR was performed with cDNA using TaqMan gene expression PCR master mix and 6-carboxyfluorescein (FAM)-MGB primers for mouse ACTA2 (assay ID, Mm00725412_s1), COL1A2 (assay ID, Mm00483888_m1), COL1A1 (assay ID, Mm00801666_g1), CTGF (assay ID, Mm01192933_g1), Vimentin (assay ID, Mm01333430_m1), FSP-1 (assay ID, Mm00803372_g1), 18S RNA (assay ID, Mm04277571_s1).

    Techniques: Activation Assay, Mouse Assay, Isolation, Expressing, Quantitative RT-PCR, Two Tailed Test

    Upregulation of hepatic stellate cell activation markers (α-SMA/COL1A1) and MMP2 by conditioned medium (CM) from HCV infected hepatocytes Primary human foreskin fibroblasts are activated in coculture by HCV replicon harboring hepatocytes. Upregulation of activation markers for HFF (α-SMA/Col1A1/CTGF) in co-culture with HCV 2a replicon were analyzed by qRT-PCR (panel A). mRNA status of activation markers were analyzed by qRT-PCR in LX2 cells upon incubation with CM from HCV infected hepatocytes (panel B). RNA from LX2 incubated with CM from mock infected hepatocytes was used as control. HCV mediated enhancement in TGF-β signaling was analyzed separately in HK293 cells transfected with a TGF-β luciferase reporter (p3TP-luc) construct, containing responsive element with minimal promoter. Cells exposed to conditioned media from HCV infected hepatocytes or neutralized with anti-TGF-β1 or anti-TGF-β2 antibodies (panel C). Luciferase activity was determined 48h post-transfection. Upregulation of activation markers in immortalized hepatic stellate cell (LX2) at mRNA level by TGF-β were analyzed by qRT-PCR. Activation markers (α-SMA/COL1A1) and MMP2 in LX-2 cells were induced at the mRNA level upon treatment with commercially available TGF-β1 (panel D) or TGF-β2 (panel E) at three different doses and analyzed by qRT-PCR. Values represent from three independent experiments ±SD. Statistical significance was analyzed using the two-tailed Student’s t test: *P

    Journal: Hepatology (Baltimore, Md.)

    Article Title: HEPATITIS C VIRUS INDUCED TUMOR INITIATING CANCER STEM-LIKE CELLS ACTIVATE STROMAL FIBROBLASTS IN XENOGRAFT TUMOR MODEL

    doi: 10.1002/hep.29346

    Figure Lengend Snippet: Upregulation of hepatic stellate cell activation markers (α-SMA/COL1A1) and MMP2 by conditioned medium (CM) from HCV infected hepatocytes Primary human foreskin fibroblasts are activated in coculture by HCV replicon harboring hepatocytes. Upregulation of activation markers for HFF (α-SMA/Col1A1/CTGF) in co-culture with HCV 2a replicon were analyzed by qRT-PCR (panel A). mRNA status of activation markers were analyzed by qRT-PCR in LX2 cells upon incubation with CM from HCV infected hepatocytes (panel B). RNA from LX2 incubated with CM from mock infected hepatocytes was used as control. HCV mediated enhancement in TGF-β signaling was analyzed separately in HK293 cells transfected with a TGF-β luciferase reporter (p3TP-luc) construct, containing responsive element with minimal promoter. Cells exposed to conditioned media from HCV infected hepatocytes or neutralized with anti-TGF-β1 or anti-TGF-β2 antibodies (panel C). Luciferase activity was determined 48h post-transfection. Upregulation of activation markers in immortalized hepatic stellate cell (LX2) at mRNA level by TGF-β were analyzed by qRT-PCR. Activation markers (α-SMA/COL1A1) and MMP2 in LX-2 cells were induced at the mRNA level upon treatment with commercially available TGF-β1 (panel D) or TGF-β2 (panel E) at three different doses and analyzed by qRT-PCR. Values represent from three independent experiments ±SD. Statistical significance was analyzed using the two-tailed Student’s t test: *P

    Article Snippet: RNA was quantified by using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized by using random hexamers and a Superscript III reverse transcriptase kit (Invitrogen, CA). qRT-PCR was performed with cDNA using TaqMan gene expression PCR master mix and 6-carboxyfluorescein (FAM)-MGB primers for mouse ACTA2 (assay ID, Mm00725412_s1), COL1A2 (assay ID, Mm00483888_m1), COL1A1 (assay ID, Mm00801666_g1), CTGF (assay ID, Mm01192933_g1), Vimentin (assay ID, Mm01333430_m1), FSP-1 (assay ID, Mm00803372_g1), 18S RNA (assay ID, Mm04277571_s1).

    Techniques: Activation Assay, Infection, Co-Culture Assay, Quantitative RT-PCR, Incubation, Transfection, Luciferase, Construct, Activity Assay, Two Tailed Test

    Assessing mineralization of 4T1 cells grown in an osteogenic cocktail within 3D collagen scaffolds. Representative images are shown at 400× and 40× magnifications and the scale bars represent 200 µm and 2000 µm respectively. (A) Positive hematoxylin and eosin (H E) staining was observed at 400× magnification by day 14 and the intensity of the hematoxylin stain increased by day 28. Some positive alizarin red S staining for calcium (red) was detected by day 14. Complete positive staining with alizarin red S and von Kossa (including toluidine blue counterstain) was observed by day 28. (B) At 40× magnification the previously described patterns of staining are confirmed and minor contraction and disintegration of the scaffolds are observed over time. (C) Assessing cell viability of 4T1 cells grown within the 3D scaffolds using an alamar blue metabolic assay. An increase in cell viability was detected between day 14 and day 28. Each time point represent the mean % reduction in alamar blue +/− SEM, n = 3, students t-test. *P = 0.0284 day 28 vs. day 14. OC (osteogenic cocktail) = with 50 µg/ml ascorbic acid and 10 mM β-glycerophosphate.

    Journal: PLoS ONE

    Article Title: Osteomimicry of Mammary Adenocarcinoma Cells In Vitro; Increased Expression of Bone Matrix Proteins and Proliferation within a 3D Collagen Environment

    doi: 10.1371/journal.pone.0041679

    Figure Lengend Snippet: Assessing mineralization of 4T1 cells grown in an osteogenic cocktail within 3D collagen scaffolds. Representative images are shown at 400× and 40× magnifications and the scale bars represent 200 µm and 2000 µm respectively. (A) Positive hematoxylin and eosin (H E) staining was observed at 400× magnification by day 14 and the intensity of the hematoxylin stain increased by day 28. Some positive alizarin red S staining for calcium (red) was detected by day 14. Complete positive staining with alizarin red S and von Kossa (including toluidine blue counterstain) was observed by day 28. (B) At 40× magnification the previously described patterns of staining are confirmed and minor contraction and disintegration of the scaffolds are observed over time. (C) Assessing cell viability of 4T1 cells grown within the 3D scaffolds using an alamar blue metabolic assay. An increase in cell viability was detected between day 14 and day 28. Each time point represent the mean % reduction in alamar blue +/− SEM, n = 3, students t-test. *P = 0.0284 day 28 vs. day 14. OC (osteogenic cocktail) = with 50 µg/ml ascorbic acid and 10 mM β-glycerophosphate.

    Article Snippet: Alamar Blue Cell viability was monitored for cells grown in 3D collagen scaffolds using an alamar blue® metabolic assay according to the manufacturer’s instructions (Invitrogen, California, USA).

    Techniques: Staining, Metabolic Assay