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fluidigm 3169013b 147sm anti cd20 clone 2h7 fluidigm
3169013b 147sm Anti Cd20 Clone 2h7 Fluidigm, supplied by fluidigm, used in various techniques. Bioz Stars score: 94/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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a , Trial schema of the BC-APPS1 study. A VAB was collected in the luteal phase (baseline), and repeated in the opposite breast after 12 weeks of UA (5 mg daily). AFM, atomic force microscopy; IF, immunofluorescence; IHC, immunohistochemistry; IMC, imaging mass cytometry. The trial schema was created using BioRender ( https://biorender.com ). b , Percentage of Ki67-positive cells in 24 paired breast tissue samples before (baseline) and after (post-treatment) 3 months of UA therapy. Representative staining is shown. c , Proportion of epithelial area per lobule area before (baseline) and after (post-treatment) 3 months of UA therapy ( n = 19 tissue pairs). Examples of lobule epithelial areas (green outlines) are shown. d , Flow cytometry analysis of luminal mature (LM; CD49f − EpCAM + ), luminal progenitor (LP; CD49f + EpCAM + ), basal (BA; CD49f + EpCAM −/low ) and stromal (S; CD49f − EpCAM − ) cells. The graph shows the percentage of epithelial populations (LP, LM and BA) in 17 tissue pairs. NS, not significant. e , Percentage of luminal, mixed or basal colonies in 18 breast tissue sample pairs before and after UA therapy. Representative examples of clonogenic assay colonies are shown above. f , MFE data expressed as a percentage for 19 tissue pairs. Horizontal dotted line, 0. A representative example of a mammosphere is shown above. g , Percentage of SOX9 and Ki67 double positive cells in eight tissue pairs quantified by immunofluorescence. The arrow in the representative images above indicates a cell expressing both SOX9 and Ki67. In all plots, boxplot centre lines represent median values and box bounds indicate the 25th and 75th percentiles, with connecting lines between paired data points. P values were calculated with two-sided Wilcoxon matched-pairs signed-rank test ( b – g ). Scale bars, 50 μm ( b , c , e , f ) and 10 μm ( g ).

Journal: Nature

Article Title: Anti-progestin therapy targets hallmarks of breast cancer risk

doi: 10.1038/s41586-025-09684-7

Figure Lengend Snippet: a , Trial schema of the BC-APPS1 study. A VAB was collected in the luteal phase (baseline), and repeated in the opposite breast after 12 weeks of UA (5 mg daily). AFM, atomic force microscopy; IF, immunofluorescence; IHC, immunohistochemistry; IMC, imaging mass cytometry. The trial schema was created using BioRender ( https://biorender.com ). b , Percentage of Ki67-positive cells in 24 paired breast tissue samples before (baseline) and after (post-treatment) 3 months of UA therapy. Representative staining is shown. c , Proportion of epithelial area per lobule area before (baseline) and after (post-treatment) 3 months of UA therapy ( n = 19 tissue pairs). Examples of lobule epithelial areas (green outlines) are shown. d , Flow cytometry analysis of luminal mature (LM; CD49f − EpCAM + ), luminal progenitor (LP; CD49f + EpCAM + ), basal (BA; CD49f + EpCAM −/low ) and stromal (S; CD49f − EpCAM − ) cells. The graph shows the percentage of epithelial populations (LP, LM and BA) in 17 tissue pairs. NS, not significant. e , Percentage of luminal, mixed or basal colonies in 18 breast tissue sample pairs before and after UA therapy. Representative examples of clonogenic assay colonies are shown above. f , MFE data expressed as a percentage for 19 tissue pairs. Horizontal dotted line, 0. A representative example of a mammosphere is shown above. g , Percentage of SOX9 and Ki67 double positive cells in eight tissue pairs quantified by immunofluorescence. The arrow in the representative images above indicates a cell expressing both SOX9 and Ki67. In all plots, boxplot centre lines represent median values and box bounds indicate the 25th and 75th percentiles, with connecting lines between paired data points. P values were calculated with two-sided Wilcoxon matched-pairs signed-rank test ( b – g ). Scale bars, 50 μm ( b , c , e , f ) and 10 μm ( g ).

Article Snippet: The antibodies used included α-smooth muscle actin (clone 1A4, 141Pr), E-cadherin (clone 24E10, 158Gd), Ki67 (clone B56, 168Er) and collagen I (polyclonal, 169Tm) from Standard BioTools (201508, Maxpar Human Immuno-oncology IMC panel kit), as well as fibronectin (polyclonal, 149Sm; ab23750, Abcam), collagen VI (polyclonal, 160Gd; ab6588, Abcam) and SOX9 (clone EPR14335 , 147Sm; 3147022D, Standard BioTools).

Techniques: Microscopy, Immunofluorescence, Immunohistochemistry, Imaging, Mass Cytometry, Staining, Flow Cytometry, Clonogenic Assay, Expressing

A) Measurement of serum progesterone levels before (baseline) and after (post-treatment) 3 months of ulipristal acetate (N = 24 pairs). B) Average area of acinar structures within lobules before (baseline) and after (post-treatment) 3 months of UA therapy (N = 17 tissue pairs). C) Number of acinar structures per area (µm 2 ) of lobules before (baseline) and after (post-treatment) 3 months of UA therapy (N = 19 tissue pairs). D) Gating strategy used to assess breast epithelial cell populations via flow cytometry. Live cell-sized singlets were selected from samples lineage depleted and stained with EpCAM and CD49f antibodies. E) Representative examples of luminal, mixed and basal colonies formed in the clonogenic assay are shown. Scale bar = 50 μm. N = 18 tissue pairs. F) Mammosphere formation efficiency (MFE %) data for baseline cell suspensions in the presence of onapristone (100 nM) versus control (DMSO). N = 14 baseline tissue. G) Mammosphere formation efficiency (MFE %) data for baseline cell suspensions in the presence of ulipristal acetate (2 nM) versus control (ethanol). N = 6 baseline tissue. H) Percentage of SOX9+ cells in pairs of tissue quantified by immunofluorescence. N = 10 tissue pairs. Box plots centre lines represent median values and box bounds indicate the 25th and 75th percentiles, with connecting lines between paired data points; p values are calculated with two-sided Wilcoxon matched-pairs signed rank test (A-C, F-H).

Journal: Nature

Article Title: Anti-progestin therapy targets hallmarks of breast cancer risk

doi: 10.1038/s41586-025-09684-7

Figure Lengend Snippet: A) Measurement of serum progesterone levels before (baseline) and after (post-treatment) 3 months of ulipristal acetate (N = 24 pairs). B) Average area of acinar structures within lobules before (baseline) and after (post-treatment) 3 months of UA therapy (N = 17 tissue pairs). C) Number of acinar structures per area (µm 2 ) of lobules before (baseline) and after (post-treatment) 3 months of UA therapy (N = 19 tissue pairs). D) Gating strategy used to assess breast epithelial cell populations via flow cytometry. Live cell-sized singlets were selected from samples lineage depleted and stained with EpCAM and CD49f antibodies. E) Representative examples of luminal, mixed and basal colonies formed in the clonogenic assay are shown. Scale bar = 50 μm. N = 18 tissue pairs. F) Mammosphere formation efficiency (MFE %) data for baseline cell suspensions in the presence of onapristone (100 nM) versus control (DMSO). N = 14 baseline tissue. G) Mammosphere formation efficiency (MFE %) data for baseline cell suspensions in the presence of ulipristal acetate (2 nM) versus control (ethanol). N = 6 baseline tissue. H) Percentage of SOX9+ cells in pairs of tissue quantified by immunofluorescence. N = 10 tissue pairs. Box plots centre lines represent median values and box bounds indicate the 25th and 75th percentiles, with connecting lines between paired data points; p values are calculated with two-sided Wilcoxon matched-pairs signed rank test (A-C, F-H).

Techniques: Flow Cytometry, Staining, Clonogenic Assay, Control, Immunofluorescence

a , Lobular epithelium and peri-lobular stroma (within 25 μm of the observable edge of the epithelium) were laser capture microdissected from haematoxylin and eosin-stained paired tissue sections before (baseline) and after (post-treatment) UA treatment. A representative example of undissected tissue (left) and tissue after laser ablation (right) is shown. n = 4 tissue pairs. Scale bar, 100 μm. b , Venn diagram representing the distribution of the total proteins detected (1,519) in the four participants (P06, P12, P17 and P31) used for LCM proteomics. c , Volcano plot shows differential protein abundance analysis following UA treatment. Matrisome (structural ECM or ECM-modifying) proteins among the significantly altered proteins are colour coded according to their respective subcategories. d , Gene set enrichment analysis of LCM proteomics data using the Reactome Pathways reference set, showing pathways significantly altered by UA treatment. e , Heatmap of the 27 matrisome proteins identified as significantly differentially abundant after UA treatment. ECM proteins are grouped by their structural and functional properties. f , Imaging mass cytometry was performed on paired tissue sections before (baseline) and after (post-treatment) UA treatment. Representative images show staining with metal-conjugated antibodies to E-cadherin, SOX9 and collagen VI. Nuclei were visualized using a metal-tagged DNA intercalator. The yellow boxes indicate regions corresponding to the zoomed-in inserts. n = 8 tissue pairs. Scale bars, 100 µm and 10 µm (insets). g , Single-cell neighbourhood analysis of pericellular collagen VI abundance in SOX9 high and SOX9 low cell populations across paired BC-APPS1 samples at baseline (B) and post-treatment (PT) timepoints. Tissue images were segmented into single-cell objects, and cells were classified based on expression of specific markers. Analysis was performed on E-cadherin + cells classified as either SOX9 high or SOX9 low . For each selected cell, collagen VI staining intensity was quantified within a 10-µm radius. Scale bar, 100 µm. Boxplot centre lines represent median values, box bounds indicate the 25th and 75th percentiles, and whiskers denote minimum and maximum values. Statistical analysis was performed using a repeated measure one-way analysis of variance (ANOVA) followed by Sidak’s multiple comparisons test. n = 8 tissue pairs.

Journal: Nature

Article Title: Anti-progestin therapy targets hallmarks of breast cancer risk

doi: 10.1038/s41586-025-09684-7

Figure Lengend Snippet: a , Lobular epithelium and peri-lobular stroma (within 25 μm of the observable edge of the epithelium) were laser capture microdissected from haematoxylin and eosin-stained paired tissue sections before (baseline) and after (post-treatment) UA treatment. A representative example of undissected tissue (left) and tissue after laser ablation (right) is shown. n = 4 tissue pairs. Scale bar, 100 μm. b , Venn diagram representing the distribution of the total proteins detected (1,519) in the four participants (P06, P12, P17 and P31) used for LCM proteomics. c , Volcano plot shows differential protein abundance analysis following UA treatment. Matrisome (structural ECM or ECM-modifying) proteins among the significantly altered proteins are colour coded according to their respective subcategories. d , Gene set enrichment analysis of LCM proteomics data using the Reactome Pathways reference set, showing pathways significantly altered by UA treatment. e , Heatmap of the 27 matrisome proteins identified as significantly differentially abundant after UA treatment. ECM proteins are grouped by their structural and functional properties. f , Imaging mass cytometry was performed on paired tissue sections before (baseline) and after (post-treatment) UA treatment. Representative images show staining with metal-conjugated antibodies to E-cadherin, SOX9 and collagen VI. Nuclei were visualized using a metal-tagged DNA intercalator. The yellow boxes indicate regions corresponding to the zoomed-in inserts. n = 8 tissue pairs. Scale bars, 100 µm and 10 µm (insets). g , Single-cell neighbourhood analysis of pericellular collagen VI abundance in SOX9 high and SOX9 low cell populations across paired BC-APPS1 samples at baseline (B) and post-treatment (PT) timepoints. Tissue images were segmented into single-cell objects, and cells were classified based on expression of specific markers. Analysis was performed on E-cadherin + cells classified as either SOX9 high or SOX9 low . For each selected cell, collagen VI staining intensity was quantified within a 10-µm radius. Scale bar, 100 µm. Boxplot centre lines represent median values, box bounds indicate the 25th and 75th percentiles, and whiskers denote minimum and maximum values. Statistical analysis was performed using a repeated measure one-way analysis of variance (ANOVA) followed by Sidak’s multiple comparisons test. n = 8 tissue pairs.

Techniques: Staining, Quantitative Proteomics, Functional Assay, Imaging, Mass Cytometry, Expressing

A) Heatmap of the 65 proteins identified as significantly differentially abundant after UA treatment. B) Representative imaging mass cytometry images showing staining with metal-conjugated antibodies against E-Cadherin (E-Cad; luminal cell marker), α-smooth muscle actin (SMA; basal cell marker) and Collagen I (Col-I; stromal marker). E-Cad and SMA markers were used to delineate the epithelial regions and define the peri-epithelial stroma (+25 µm from epithelia). Box plots compare Col-I, Col-VI and FN1 mean intensity in epithelia and peri-epithelial stroma of paired tissue sections before (B) and after (PT) UA treatment. Box plots centre lines represent median values, box bounds indicate the 25th and 75th percentiles, and whiskers denote minima and maxima values. Statistical analysis was performed using two-sided Wilcoxon matched pairs signed rank test. N = 8 tissue pairs. C) Single-cell neighbourhood analysis of pericellular collagen I (left hand panel) and fibronectin (right hand panel) abundance for SOX9 high and SOX9 low populations across paired BC-APPS1 samples at baseline (B) and post treatment (PT) timepoints. Single-cell neighbourhood analysis was performed as described in Fig. . For each selected cell, Collagen-I or fibronectin staining intensity was quantified within a 10 µm radius. Box plots centre lines represent median values, box bounds indicate the 25th and 75th percentiles, and whiskers denote minima and maxima values. Statistical analysis was performed using a repeated measure one-way ANOVA followed by Sidak’s multiple comparisons test. N = 8 tissue pairs. D) The percentage of Ki67+ cells in epithelial SOX9 high and SOX9 low cells populations were calculated across the samples described in Extended Data Fig. 11c. Box plots centre lines represent median values, box bounds indicate the 25 th and 75 th percentiles, and whiskers denote minima and maxima values. Statistical analysis was performed using repeated measure one-way ANOVA followed by Sidak’s multiple comparisons test. N = 8 tissue pairs.

Journal: Nature

Article Title: Anti-progestin therapy targets hallmarks of breast cancer risk

doi: 10.1038/s41586-025-09684-7

Figure Lengend Snippet: A) Heatmap of the 65 proteins identified as significantly differentially abundant after UA treatment. B) Representative imaging mass cytometry images showing staining with metal-conjugated antibodies against E-Cadherin (E-Cad; luminal cell marker), α-smooth muscle actin (SMA; basal cell marker) and Collagen I (Col-I; stromal marker). E-Cad and SMA markers were used to delineate the epithelial regions and define the peri-epithelial stroma (+25 µm from epithelia). Box plots compare Col-I, Col-VI and FN1 mean intensity in epithelia and peri-epithelial stroma of paired tissue sections before (B) and after (PT) UA treatment. Box plots centre lines represent median values, box bounds indicate the 25th and 75th percentiles, and whiskers denote minima and maxima values. Statistical analysis was performed using two-sided Wilcoxon matched pairs signed rank test. N = 8 tissue pairs. C) Single-cell neighbourhood analysis of pericellular collagen I (left hand panel) and fibronectin (right hand panel) abundance for SOX9 high and SOX9 low populations across paired BC-APPS1 samples at baseline (B) and post treatment (PT) timepoints. Single-cell neighbourhood analysis was performed as described in Fig. . For each selected cell, Collagen-I or fibronectin staining intensity was quantified within a 10 µm radius. Box plots centre lines represent median values, box bounds indicate the 25th and 75th percentiles, and whiskers denote minima and maxima values. Statistical analysis was performed using a repeated measure one-way ANOVA followed by Sidak’s multiple comparisons test. N = 8 tissue pairs. D) The percentage of Ki67+ cells in epithelial SOX9 high and SOX9 low cells populations were calculated across the samples described in Extended Data Fig. 11c. Box plots centre lines represent median values, box bounds indicate the 25 th and 75 th percentiles, and whiskers denote minima and maxima values. Statistical analysis was performed using repeated measure one-way ANOVA followed by Sidak’s multiple comparisons test. N = 8 tissue pairs.

Techniques: Imaging, Mass Cytometry, Staining, Marker

a , Real-time PCR gene expression of TNFSF11 , KIT and SOX9 in breast tissue microstructures from women at increased cancer risk, cultured in ‘soft’ and ‘stiff’ hydrogels. Data are shown as mean fold change ± s.d., with individual points. n = 6 breast samples. b , KIT and SOX9 protein in breast microstructures (sample 1989N) cultured in soft (S) and stiff (ST) hydrogels, treated with UA (2 nM) or onapristone (ON; 100 nM). Densitometry normalized to β-actin is shown above the bands. n = 3 breast samples. c , MFE after culture in soft and stiff hydrogels with UA (2 nM) or ON (100 nM). Data are shown as mean fold change ± s.d., with individual points. n = 6 breast samples. d , Collagen coherency was assessed in peri-lobular regions (three lobules per sample) with representative PSR-stained sections shown at baseline and post-treatment. The ellipse indicates fibre alignment: examples of aligned (baseline) and non-aligned (post-treatment) collagen are shown in the insets. The graph shows mean collagen coherency for n = 22 paired samples. Scale bars, 100 μm. e , Reduced modulus of peri-lobular regions at baseline (B) and post-treatment (PT) measured by AFM indentation. At least three 100 μm 2 regions per sample were measured as shown in the representative images. n = 4 tissue pairs. Scale bars, 100 μm. f , MRI annotation in ITK-snap: black denotes the background, opaque red indicates fatty tissue, and bright red shows the fibroglandular tissue. The FGV percentage was calculated by dividing the number of fibroglandular pixels by the total number of fibroglandular and fat pixels across slices. n = 12 paired MRI scans. Scale bars, 1 cm. g , Percentage of Ki67 + cells before treatment and post-treatment stratified by mammographic density. Participants were grouped using Volpara density grades to approximate BI-RADS categories (A/B denotes low MD, n = 6 tissue pairs; C/D indicates high MD, n = 17 tissue pairs). h , Heatmap of whole-tissue RNA-seq showing the differentially expressed genes between high MD (BI-RADS C/D; dark grey) and low MD (BI-RADS A/B; light grey) breast tissue at baseline ( n = 9; FC > 3, P < 0.05). VST, variance-stabilizing transformation. i , Illustration shows that progesterone paracrine signalling regulates luminal progenitor/LASP (SOX9 + ) cells and fibroblasts, driving ECM remodelling and stiffness. Stiffness amplifies PR signalling, establishing a feedback loop. Anti-progestins disrupt this by inhibiting luminal cell-derived ligands (for example, WNT5A), lowering fibroblast collagen (for example, COL6A3), decreasing stiffness and reducing luminal progenitor/LASP cells. Boxplot centre lines represent median values and box bounds indicate the 25th and 75th percentiles ( d – g ), with connecting lines between paired data points ( d , f , g ) or whiskers denoting minimum and maximum values ( e ). P values were calculated with two-sided Wilcoxon matched-pairs test ( a , c , d , f , g ) or two-sided Student’s t -test ( e ).

Journal: Nature

Article Title: Anti-progestin therapy targets hallmarks of breast cancer risk

doi: 10.1038/s41586-025-09684-7

Figure Lengend Snippet: a , Real-time PCR gene expression of TNFSF11 , KIT and SOX9 in breast tissue microstructures from women at increased cancer risk, cultured in ‘soft’ and ‘stiff’ hydrogels. Data are shown as mean fold change ± s.d., with individual points. n = 6 breast samples. b , KIT and SOX9 protein in breast microstructures (sample 1989N) cultured in soft (S) and stiff (ST) hydrogels, treated with UA (2 nM) or onapristone (ON; 100 nM). Densitometry normalized to β-actin is shown above the bands. n = 3 breast samples. c , MFE after culture in soft and stiff hydrogels with UA (2 nM) or ON (100 nM). Data are shown as mean fold change ± s.d., with individual points. n = 6 breast samples. d , Collagen coherency was assessed in peri-lobular regions (three lobules per sample) with representative PSR-stained sections shown at baseline and post-treatment. The ellipse indicates fibre alignment: examples of aligned (baseline) and non-aligned (post-treatment) collagen are shown in the insets. The graph shows mean collagen coherency for n = 22 paired samples. Scale bars, 100 μm. e , Reduced modulus of peri-lobular regions at baseline (B) and post-treatment (PT) measured by AFM indentation. At least three 100 μm 2 regions per sample were measured as shown in the representative images. n = 4 tissue pairs. Scale bars, 100 μm. f , MRI annotation in ITK-snap: black denotes the background, opaque red indicates fatty tissue, and bright red shows the fibroglandular tissue. The FGV percentage was calculated by dividing the number of fibroglandular pixels by the total number of fibroglandular and fat pixels across slices. n = 12 paired MRI scans. Scale bars, 1 cm. g , Percentage of Ki67 + cells before treatment and post-treatment stratified by mammographic density. Participants were grouped using Volpara density grades to approximate BI-RADS categories (A/B denotes low MD, n = 6 tissue pairs; C/D indicates high MD, n = 17 tissue pairs). h , Heatmap of whole-tissue RNA-seq showing the differentially expressed genes between high MD (BI-RADS C/D; dark grey) and low MD (BI-RADS A/B; light grey) breast tissue at baseline ( n = 9; FC > 3, P < 0.05). VST, variance-stabilizing transformation. i , Illustration shows that progesterone paracrine signalling regulates luminal progenitor/LASP (SOX9 + ) cells and fibroblasts, driving ECM remodelling and stiffness. Stiffness amplifies PR signalling, establishing a feedback loop. Anti-progestins disrupt this by inhibiting luminal cell-derived ligands (for example, WNT5A), lowering fibroblast collagen (for example, COL6A3), decreasing stiffness and reducing luminal progenitor/LASP cells. Boxplot centre lines represent median values and box bounds indicate the 25th and 75th percentiles ( d – g ), with connecting lines between paired data points ( d , f , g ) or whiskers denoting minimum and maximum values ( e ). P values were calculated with two-sided Wilcoxon matched-pairs test ( a , c , d , f , g ) or two-sided Student’s t -test ( e ).

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Cell Culture, Staining, RNA Sequencing, Transformation Assay, Derivative Assay

A-B) C-KIT and SOX9 protein detection in normal breast microstructures cultured in collagen-mimetic hydrogels under ‘soft’ (S) and ‘stiff’ (ST) conditions, treated for 7 days with: A) Ulipristal Acetate (UA, 2 nM), or B) Onapristone (ON, 100 nM). β-actin was used as a reference for the loading control. Densitometry quantification, normalised to β-actin, is shown at the top of each band. N = 3 breast samples. C) Heatmap from whole tissue RNA-Seq gene expression analysis showing all the differentially expressed genes between high MD (BI-RADS C/D, dark grey) and low MD (BI-RADS A/B, light grey) breast tissue at BC-APPS1 baseline (n = 9; p < 0.05). VST - Variance Stabilising Transformation. D) Correlation (two-sided test) between VBD percentage and gene expression of CXCL13 and TNFSF11 in baseline breast tissue of BC-APPS1 participants. N = 9 baseline tissue.

Journal: Nature

Article Title: Anti-progestin therapy targets hallmarks of breast cancer risk

doi: 10.1038/s41586-025-09684-7

Figure Lengend Snippet: A-B) C-KIT and SOX9 protein detection in normal breast microstructures cultured in collagen-mimetic hydrogels under ‘soft’ (S) and ‘stiff’ (ST) conditions, treated for 7 days with: A) Ulipristal Acetate (UA, 2 nM), or B) Onapristone (ON, 100 nM). β-actin was used as a reference for the loading control. Densitometry quantification, normalised to β-actin, is shown at the top of each band. N = 3 breast samples. C) Heatmap from whole tissue RNA-Seq gene expression analysis showing all the differentially expressed genes between high MD (BI-RADS C/D, dark grey) and low MD (BI-RADS A/B, light grey) breast tissue at BC-APPS1 baseline (n = 9; p < 0.05). VST - Variance Stabilising Transformation. D) Correlation (two-sided test) between VBD percentage and gene expression of CXCL13 and TNFSF11 in baseline breast tissue of BC-APPS1 participants. N = 9 baseline tissue.

Techniques: Cell Culture, Control, RNA Sequencing, Gene Expression, Transformation Assay

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