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biotinylated anti cxcl12 antibody (R&D Systems)

Name: biotinylated anti cxcl12 antibody
Brand: R&D Systems
Rating: 93 (51 reviews)

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R&D Systems biotinylated anti cxcl12 antibody
Biotinylated Anti Cxcl12 Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 51 article reviews

biotinylated anti cxcl12 antibody - by Bioz Stars, 2026-02

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Optimization of the ISTAMPA method for detection of CXCL12 proteoforms. A Principle of top-down MS/MS fragmentation of CXCL12(1-68) or CXCL12(3-68) through low-energy collision-induced dissociation (CID). Fragmentation occurs selectively at the acid-labile bond between Asp52 (D) and Pro53 (P) after isolation of the (B) precursor ion with the highest intensity in the single MS spectrum [mass-to-charge (m/z) value of 885.4 (9 +) for intact CXCL12(1-68) or 860.3 (9 +) for NH2-terminally truncated CXCL12(3-68)]. C Extracted ion chromatogram (EIC) and MS/MS spectrum for CXCL12(1-68) and CXCL12(3-68). The EIC shows the intensity of the signature fragment ions (with mass accuracy of ± 0.5 mass unit) during chemokine elution from the nano-RP-UPLC column at t = 22–24 min. CID fragmentation generates two specific fragments per CXCL12 proteoform, with each two characteristic fragment ions (indicated by their m/z value and charge on the spectra). The red diamond in the MS/MS spectra indicates the m/z of the precursor ion selected for CID fragmentation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Optimization of the ISTAMPA method for detection of CXCL12 proteoforms. A Principle of top-down MS/MS fragmentation of CXCL12(1-68) or CXCL12(3-68) through low-energy collision-induced dissociation (CID). Fragmentation occurs selectively at the acid-labile bond between Asp52 (D) and Pro53 (P) after isolation of the (B) precursor ion with the highest intensity in the single MS spectrum [mass-to-charge (m/z) value of 885.4 (9 +) for intact CXCL12(1-68) or 860.3 (9 +) for NH2-terminally truncated CXCL12(3-68)]. C Extracted ion chromatogram (EIC) and MS/MS spectrum for CXCL12(1-68) and CXCL12(3-68). The EIC shows the intensity of the signature fragment ions (with mass accuracy of ± 0.5 mass unit) during chemokine elution from the nano-RP-UPLC column at t = 22–24 min. CID fragmentation generates two specific fragments per CXCL12 proteoform, with each two characteristic fragment ions (indicated by their m/z value and charge on the spectra). The red diamond in the MS/MS spectra indicates the m/z of the precursor ion selected for CID fragmentation

Article Snippet: Afterward, 0.25 μg/mL biotinylated polyclonal rabbit anti-human CXCL12 antibodies (#500-P87ABT; PeproTech) with HRP-conjugated streptavidin (R&D Systems) were employed for detection.

Techniques: Tandem Mass Spectroscopy, Isolation

Proteolytic processing of CXCL12 in BAL fluid. Recombinant human CXCL12(1-68) (250 ng) was spiked in 20 µL of BAL fluid from (A–D) COVID-19 patients, grouped according to the proteolytic activity in their BAL fluid (n = 9–12), (E,F) stable lung transplantation patients (n = 6), or (G,H) the non-affected lung of lung cancer patients (n = 4) and incubated for 3 h at 37 °C. A,C,E,G Immediately after spiking (without incubation) and (B,D,F,H) after 3 h of incubation at 37 °C, CXCL12 proteolysis was determined by ISTAMPA. ISTAMPA included a 30-min pre-purification at room temperature (RT) of the CXCL12 proteoforms before nano-LC-MS/MS analysis. Only those proteoforms that were detected at least once were included in the graphs. Total CXCL12 was below the detection limit in 4 out of the 12 samples with high proteolytic activity after 3 h of incubation. CXCL12 proteoforms were presented according to their relative abundance in the sample, expressed as percentage of total CXCL12. Data are shown as dots, representing individual patient samples, in a bar graph, indicating the mean ± SEM

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Proteolytic processing of CXCL12 in BAL fluid. Recombinant human CXCL12(1-68) (250 ng) was spiked in 20 µL of BAL fluid from (A–D) COVID-19 patients, grouped according to the proteolytic activity in their BAL fluid (n = 9–12), (E,F) stable lung transplantation patients (n = 6), or (G,H) the non-affected lung of lung cancer patients (n = 4) and incubated for 3 h at 37 °C. A,C,E,G Immediately after spiking (without incubation) and (B,D,F,H) after 3 h of incubation at 37 °C, CXCL12 proteolysis was determined by ISTAMPA. ISTAMPA included a 30-min pre-purification at room temperature (RT) of the CXCL12 proteoforms before nano-LC-MS/MS analysis. Only those proteoforms that were detected at least once were included in the graphs. Total CXCL12 was below the detection limit in 4 out of the 12 samples with high proteolytic activity after 3 h of incubation. CXCL12 proteoforms were presented according to their relative abundance in the sample, expressed as percentage of total CXCL12. Data are shown as dots, representing individual patient samples, in a bar graph, indicating the mean ± SEM

Techniques: Recombinant, Activity Assay, Transplantation Assay, Incubation, Purification, Liquid Chromatography with Mass Spectroscopy

Identification of CXCL12-cleaving proteases in COVID-19 BAL fluid. Recombinant human CXCL12(1-68) (250 ng) was spiked in 20 µL of BAL fluid from COVID-19 patients, in the absence (“not treated”) or presence (“treated”) of different protease inhibitors. After 3 h of incubation at 37 °C, CXCL12 proteolysis was determined by ISTAMPA. Different BAL fluid samples were selected per inhibitor according to the proteolytic processing that was observed with untreated samples. A Neutrophil elastase inhibition with sivelestat in high proteolytic activity (HPA) BAL fluid (n = 4). Total CXCL12 was below the detection limit in two untreated samples. B Serine protease inhibition with AEBSF in HPA BAL fluid (n = 2). Total CXCL12 was below the detection limit in both untreated samples. C Metalloprotease inhibition with EDTA in low proteolytic activity (LPA) BAL fluid samples (n = 3). D CD26 inhibition with sitagliptin in LPA BAL fluid samples (n = 3). E Cysteine protease inhibition with E-64 in LPA BAL fluid samples (n = 2). Only those proteoforms that were detected at least once were included in the graphs. CXCL12 proteoforms were presented according to their relative abundance in the sample, expressed as percentage of total CXCL12. Data are shown as dots, representing individual patient samples, in a bar graph, indicating the mean ± SEM

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Identification of CXCL12-cleaving proteases in COVID-19 BAL fluid. Recombinant human CXCL12(1-68) (250 ng) was spiked in 20 µL of BAL fluid from COVID-19 patients, in the absence (“not treated”) or presence (“treated”) of different protease inhibitors. After 3 h of incubation at 37 °C, CXCL12 proteolysis was determined by ISTAMPA. Different BAL fluid samples were selected per inhibitor according to the proteolytic processing that was observed with untreated samples. A Neutrophil elastase inhibition with sivelestat in high proteolytic activity (HPA) BAL fluid (n = 4). Total CXCL12 was below the detection limit in two untreated samples. B Serine protease inhibition with AEBSF in HPA BAL fluid (n = 2). Total CXCL12 was below the detection limit in both untreated samples. C Metalloprotease inhibition with EDTA in low proteolytic activity (LPA) BAL fluid samples (n = 3). D CD26 inhibition with sitagliptin in LPA BAL fluid samples (n = 3). E Cysteine protease inhibition with E-64 in LPA BAL fluid samples (n = 2). Only those proteoforms that were detected at least once were included in the graphs. CXCL12 proteoforms were presented according to their relative abundance in the sample, expressed as percentage of total CXCL12. Data are shown as dots, representing individual patient samples, in a bar graph, indicating the mean ± SEM

Techniques: Recombinant, Incubation, Inhibition, Activity Assay

Proteolytic processing of CXCL12 in plasma. Recombinant human CXCL12(1–68) (250 ng) was spiked in 20 µL of platelet-free plasma from (A,B) COVID-19 patients (n = 7) or (C,D) healthy controls (n = 7) and incubated for 3 h at 37 °C. A,C Immediately after spiking (without incubation) and (B,D) after 3 h of incubation at 37 °C, CXCL12 proteolysis was determined by ISTAMPA. ISTAMPA included a 30-min pre-purification at room temperature (RT) of the CXCL12 proteoforms before nano-LC-MS/MS analysis. Only those proteoforms that were detected at least once were included in the graphs. CXCL12 proteoforms were presented according to their relative abundance in the sample, expressed as percentage of total CXCL12. E,F CD26 activity was determined using a colorimetric assay in these plasma samples and in BAL fluids from stable lung transplantation patients (n = 6) and COVID-19 patients (n = 21), separated according to (F) high (n = 12) or low (n = 9) proteolytic activity. Data are shown as dots, representing individual patient samples, in a bar graph, indicating the mean ± SEM. Statistical analysis was performed by Brown–Forsythe and Welch ANOVA tests with a Dunnett’s T3 multiple comparisons test and an unpaired t test. *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Proteolytic processing of CXCL12 in plasma. Recombinant human CXCL12(1–68) (250 ng) was spiked in 20 µL of platelet-free plasma from (A,B) COVID-19 patients (n = 7) or (C,D) healthy controls (n = 7) and incubated for 3 h at 37 °C. A,C Immediately after spiking (without incubation) and (B,D) after 3 h of incubation at 37 °C, CXCL12 proteolysis was determined by ISTAMPA. ISTAMPA included a 30-min pre-purification at room temperature (RT) of the CXCL12 proteoforms before nano-LC-MS/MS analysis. Only those proteoforms that were detected at least once were included in the graphs. CXCL12 proteoforms were presented according to their relative abundance in the sample, expressed as percentage of total CXCL12. E,F CD26 activity was determined using a colorimetric assay in these plasma samples and in BAL fluids from stable lung transplantation patients (n = 6) and COVID-19 patients (n = 21), separated according to (F) high (n = 12) or low (n = 9) proteolytic activity. Data are shown as dots, representing individual patient samples, in a bar graph, indicating the mean ± SEM. Statistical analysis was performed by Brown–Forsythe and Welch ANOVA tests with a Dunnett’s T3 multiple comparisons test and an unpaired t test. *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001

Techniques: Clinical Proteomics, Recombinant, Incubation, Purification, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Colorimetric Assay, Transplantation Assay

Proteolytic degradation of CXCL12 in BAL fluid and plasma. Recombinant human CXCL12(1-68) (250 ng) was spiked in 20 µL of (A, B) BAL fluid from COVID-19 (n = 17) or (C) stable lung transplantation patients (n = 6); platelet-free plasma from (D) COVID-19 patients (n = 7) or (E) healthy controls (n = 7); or (F) BAL fluid from COVID-19 patients in the absence (“not treated”) or presence (“treated”) of protease inhibitors (n = 2–4) and incubated for 3 h at 37 °C. A–E Immediately after spiking (at t = 0 min) and after 3 h of incubation at 37 °C, total CXCL12 levels were determined by ELISA. In (B), the fold change in CXCL12 concentration after 3 h of incubation is shown in all COVID-19 BAL fluid samples (n = 17) and separated in high (n = 8) and low proteolytic activity samples (n = 9). In (F), the CXCL12 levels after 3 h of incubation are displayed. The dotted line represents the maximal value of 250 ng CXCL12 that was spiked in the samples. The dashed line represents the lower detection limit of the ELISA assay. Samples were tested in quadruplicate, i.e., at two different dilutions and with two different antibody combinations. Data are shown as dots, representing individual patient samples, in a bar graph indicating median ± interquartile range. Statistical analysis for (A, C–E) paired samples was performed by Wilcoxon matched-pairs signed rank tests and (B) a Mann–Whitney U test for unpaired samples.*p ≤ 0.05; ****p ≤ 0.0001

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Proteolytic degradation of CXCL12 in BAL fluid and plasma. Recombinant human CXCL12(1-68) (250 ng) was spiked in 20 µL of (A, B) BAL fluid from COVID-19 (n = 17) or (C) stable lung transplantation patients (n = 6); platelet-free plasma from (D) COVID-19 patients (n = 7) or (E) healthy controls (n = 7); or (F) BAL fluid from COVID-19 patients in the absence (“not treated”) or presence (“treated”) of protease inhibitors (n = 2–4) and incubated for 3 h at 37 °C. A–E Immediately after spiking (at t = 0 min) and after 3 h of incubation at 37 °C, total CXCL12 levels were determined by ELISA. In (B), the fold change in CXCL12 concentration after 3 h of incubation is shown in all COVID-19 BAL fluid samples (n = 17) and separated in high (n = 8) and low proteolytic activity samples (n = 9). In (F), the CXCL12 levels after 3 h of incubation are displayed. The dotted line represents the maximal value of 250 ng CXCL12 that was spiked in the samples. The dashed line represents the lower detection limit of the ELISA assay. Samples were tested in quadruplicate, i.e., at two different dilutions and with two different antibody combinations. Data are shown as dots, representing individual patient samples, in a bar graph indicating median ± interquartile range. Statistical analysis for (A, C–E) paired samples was performed by Wilcoxon matched-pairs signed rank tests and (B) a Mann–Whitney U test for unpaired samples.*p ≤ 0.05; ****p ≤ 0.0001

Techniques: Clinical Proteomics, Recombinant, Transplantation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Activity Assay, MANN-WHITNEY

Western blot analysis of CXCL12 degradation in BAL fluid. One low proteolytic activity (LPA) and one high proteolytic activity (HPA) representative COVID-19 BAL fluid sample were incubated, with or without the neutrophil elastase inhibitor sivelestat and recombinant human CXCL12(1-68) before loading on the gel. An FITC-coupled anti-human CD19 rat IgG antibody was added in equal amounts to each sample before SDS-PAGE and used as loading control. Immunofluorescence was detected by polyclonal rabbit anti-human CXCL12 primary antibodies, and IRDye 680RD donkey anti-rabbit + IRDye 800CW goat anti-rat secondary antibodies, measuring fluorescence at λ = 700 nm and λ = 800 nm, respectively. The CXCL12/Light chain of rat IgG fluorescence intensity ratio is used as relative measure of residual CXCL12

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Western blot analysis of CXCL12 degradation in BAL fluid. One low proteolytic activity (LPA) and one high proteolytic activity (HPA) representative COVID-19 BAL fluid sample were incubated, with or without the neutrophil elastase inhibitor sivelestat and recombinant human CXCL12(1-68) before loading on the gel. An FITC-coupled anti-human CD19 rat IgG antibody was added in equal amounts to each sample before SDS-PAGE and used as loading control. Immunofluorescence was detected by polyclonal rabbit anti-human CXCL12 primary antibodies, and IRDye 680RD donkey anti-rabbit + IRDye 800CW goat anti-rat secondary antibodies, measuring fluorescence at λ = 700 nm and λ = 800 nm, respectively. The CXCL12/Light chain of rat IgG fluorescence intensity ratio is used as relative measure of residual CXCL12

Techniques: Western Blot, Activity Assay, Incubation, Recombinant, SDS Page, Control, Immunofluorescence, Fluorescence

Overview of the results. In the sequence of CXCL12(1-68), only the proteases likely involved in proteolytic processing of CXCL12 in COVID-19 patient samples are indicated. Abbreviations: BAL broncho-alveolar lavage, CPM/CPN carboxypeptidase M/N, HC healthy control, HPA high proteolytic activity, LPA low proteolytic activity, LTx lung transplant

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Overview of the results. In the sequence of CXCL12(1-68), only the proteases likely involved in proteolytic processing of CXCL12 in COVID-19 patient samples are indicated. Abbreviations: BAL broncho-alveolar lavage, CPM/CPN carboxypeptidase M/N, HC healthy control, HPA high proteolytic activity, LPA low proteolytic activity, LTx lung transplant

Techniques: Sequencing, Control, Activity Assay

Serial sections of normal and diseased pancreatic tissue were stained for CXCL12, CXCR4, CXCR7, and CK19. CXCL12 staining apparent in normal exocrine ducts was diminished in PDAC tissue. CXCR4 staining increased in PanIN and PDAC relative to normal ductal epithelium. CXCR7 expression was variable in normal epithelium, PanIN lesions, and PDAC. (A)Normal tissue from a single patient with healthy pancreas represents observations from 25 different normal tissues from 21 individual patients. (B)PDAC tissue from one patient represents observations from 82 different tissues from 29 different patients. 1000× magnification represents inset box at 200×. (C) Staining was quantified by blinded scoring of serial sections in relation to CK19 staining. (***) denotes P ≤0.001.

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: Serial sections of normal and diseased pancreatic tissue were stained for CXCL12, CXCR4, CXCR7, and CK19. CXCL12 staining apparent in normal exocrine ducts was diminished in PDAC tissue. CXCR4 staining increased in PanIN and PDAC relative to normal ductal epithelium. CXCR7 expression was variable in normal epithelium, PanIN lesions, and PDAC. (A)Normal tissue from a single patient with healthy pancreas represents observations from 25 different normal tissues from 21 individual patients. (B)PDAC tissue from one patient represents observations from 82 different tissues from 29 different patients. 1000× magnification represents inset box at 200×. (C) Staining was quantified by blinded scoring of serial sections in relation to CK19 staining. (***) denotes P ≤0.001.

Article Snippet: Secreted CXCL12 protein from supernatant of pancreatic cancer cells cultured in serum-free media was detected by ourpreviously established sandwich ELISA method using antibodies from R&D Systems (monoclonal mouse and human CXCL12 (MAB350) and goat anti-human CXCL12 (BAF310) .Cell surface CXCR4 or CXCR7was detected using the aforementioned antibodies (Abcam) along withFITC-conjugated secondary antibodies using our previously established method .

Techniques: Staining, Expressing

Representative 200× images of the same (A) normal or (B) pancreatic ductal adenocarcinoma (PDAC) tissues shown at 1000× magnification in . Representative serial section images of a pancreatic intestinal neoplasm (PanIN) lesion at (C) 200× or (D) 1000× magnification. Serial tissue sections were immunostained with antibodies to CK19, CXCL12, CXCR4, and CXCR7, or the isotype controls. n = 25 different normal tissues from 21 individual patients, 82 different tissues from 29 different PDAC patients, or 19 pathologically confirmed PanIN lesions.

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: Representative 200× images of the same (A) normal or (B) pancreatic ductal adenocarcinoma (PDAC) tissues shown at 1000× magnification in . Representative serial section images of a pancreatic intestinal neoplasm (PanIN) lesion at (C) 200× or (D) 1000× magnification. Serial tissue sections were immunostained with antibodies to CK19, CXCL12, CXCR4, and CXCR7, or the isotype controls. n = 25 different normal tissues from 21 individual patients, 82 different tissues from 29 different PDAC patients, or 19 pathologically confirmed PanIN lesions.

Techniques:

RT-PCR analysis revealed that (A) patient-derived pancreatic cancer cell lines (#1, #2, #3, #4) or (B) established cell lines lacked expression of CXCL12 and maintained expression of CXCR4. CXCR7 mRNA was present in 3 of 8 PDAC lines.Flow cytometric detection(C)of surface CXCR4 or CXCR7 protein expression. Cell lines treatedseven days with a concentration curve of (D) 5-aza-2-deoxycytidine (5-aza) restored expression of CXCL12.Linestreated 4 days with a concentration curve of (E) Trichostatin-A (TSA)restored CXCL12 mRNA expression in Capan2 cells.

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: RT-PCR analysis revealed that (A) patient-derived pancreatic cancer cell lines (#1, #2, #3, #4) or (B) established cell lines lacked expression of CXCL12 and maintained expression of CXCR4. CXCR7 mRNA was present in 3 of 8 PDAC lines.Flow cytometric detection(C)of surface CXCR4 or CXCR7 protein expression. Cell lines treatedseven days with a concentration curve of (D) 5-aza-2-deoxycytidine (5-aza) restored expression of CXCL12.Linestreated 4 days with a concentration curve of (E) Trichostatin-A (TSA)restored CXCL12 mRNA expression in Capan2 cells.

Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Expressing, Concentration Assay

(A) Panc1 and MiaPaCa2 cells migrated towards exogenous gradients of CXCL12 in a range from 1 nM to 1000 nM under serum-free conditions(*), (**), and (***) denote P ≤0.05, P ≤0.01, and P ≤0.001, respectively, in comparison to unstimulated cells (NS). Receptor-null HPAFII cells did not migrate in response to CXCL12. (B) CXCL12 directs Panc1 cell chemoinvasion into a three-dimensional Matrigel plug. The positive control (+) was 10% serum-containing medium. (*), (**), and (***) denote P ≤0.05, P ≤0.01, and P ≤0.001, respectively, in comparison to unstimulated cells (NS).

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: (A) Panc1 and MiaPaCa2 cells migrated towards exogenous gradients of CXCL12 in a range from 1 nM to 1000 nM under serum-free conditions(*), (**), and (***) denote P ≤0.05, P ≤0.01, and P ≤0.001, respectively, in comparison to unstimulated cells (NS). Receptor-null HPAFII cells did not migrate in response to CXCL12. (B) CXCL12 directs Panc1 cell chemoinvasion into a three-dimensional Matrigel plug. The positive control (+) was 10% serum-containing medium. (*), (**), and (***) denote P ≤0.05, P ≤0.01, and P ≤0.001, respectively, in comparison to unstimulated cells (NS).

Techniques: Positive Control

Luciferase levels in control GFP or three different (31, #2, #3) CXCL12-expressing MiaPaCa2 clones as measured by spectrophotometer (A) or IVIS-100 biophotonic imager (B). Levels of CXCL12 were measured by ELISA (C) in established PDAC cell lines as well as GFP- and CXCL12-expressing MiaPaCa2-luciferase clones. (D) CXCL12-secreted by transfected MiaPaCa2-luciferase clones #1 and #2 stimulated U937 chemotaxis. Cells treated with neutralizing antibody to CXCL12 (αL12) confirmed the specificity of U937 chemotaxis. Values in A, C, and D are mean±SEM, n = 2–3.

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: Luciferase levels in control GFP or three different (31, #2, #3) CXCL12-expressing MiaPaCa2 clones as measured by spectrophotometer (A) or IVIS-100 biophotonic imager (B). Levels of CXCL12 were measured by ELISA (C) in established PDAC cell lines as well as GFP- and CXCL12-expressing MiaPaCa2-luciferase clones. (D) CXCL12-secreted by transfected MiaPaCa2-luciferase clones #1 and #2 stimulated U937 chemotaxis. Cells treated with neutralizing antibody to CXCL12 (αL12) confirmed the specificity of U937 chemotaxis. Values in A, C, and D are mean±SEM, n = 2–3.

Techniques: Luciferase, Expressing, Clone Assay, Spectrophotometry, Enzyme-linked Immunosorbent Assay, Transfection, Chemotaxis Assay

(A) Transwell migration assays revealed significantly reduced chemotaxis of CXCL12-expressing clones (#1 and #2) compared to ligand null (GFP) cells. Attractants were serum-free media (--), 10% serum (+), or 10 nM CXCL12 (L12) in serum-free media. denote P ≤0.01 and P ≤0.001, respectively, compared to 10 nM CXCL12-stimulated GFP cells, n = 5. (B) CXCL12 re-expression diminished TGF-β (5 ng/mL)-induced chemotaxis relative to the CXCL12-null cells. (##) denotes P ≤0.01 compared to TGF-β-stimulated GFP cells, n = 4. (C) & (D) Representative images of experiments in (A) and (B) respectively. (E) CXCL12-expressing cells were significantly more adherent to tissue culture plastic compared to CXCL12-null cells. Untreated cells = (--), neutralizing antibody for CXCL12 activity = (αL12), and a positive control = 1 ng/mL of EGF (+). (##) denotes P ≤0.01 compared to 10% serum-stimulated control cells. (*), (**), and (***) denote P ≤0.05, P ≤0.01 and P ≤0.001, respectively, compared to unstimulated GFP cells, n = 7.

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: (A) Transwell migration assays revealed significantly reduced chemotaxis of CXCL12-expressing clones (#1 and #2) compared to ligand null (GFP) cells. Attractants were serum-free media (--), 10% serum (+), or 10 nM CXCL12 (L12) in serum-free media. denote P ≤0.01 and P ≤0.001, respectively, compared to 10 nM CXCL12-stimulated GFP cells, n = 5. (B) CXCL12 re-expression diminished TGF-β (5 ng/mL)-induced chemotaxis relative to the CXCL12-null cells. (##) denotes P ≤0.01 compared to TGF-β-stimulated GFP cells, n = 4. (C) & (D) Representative images of experiments in (A) and (B) respectively. (E) CXCL12-expressing cells were significantly more adherent to tissue culture plastic compared to CXCL12-null cells. Untreated cells = (--), neutralizing antibody for CXCL12 activity = (αL12), and a positive control = 1 ng/mL of EGF (+). (##) denotes P ≤0.01 compared to 10% serum-stimulated control cells. (*), (**), and (***) denote P ≤0.05, P ≤0.01 and P ≤0.001, respectively, compared to unstimulated GFP cells, n = 7.

Techniques: Migration, Chemotaxis Assay, Expressing, Clone Assay, Activity Assay, Positive Control

(A) Representative bioluminescence images of mice xenografted with GFP- or CXCL12-expressing cells at implantation (Day 0) or study endpoint (Day 28). (B) Whole-body in vivo radiance over time of GFP- and CXCL12-mice. (C) Ex vivo radiance of excised spleen (C), reflecting tumor cells at the site of injection, or the metastatic destination (D), reflecting decreased hepatic metastasis of CXCL12-expressing cells relative to GFP-cells. (**) denotes P ≤0.01, n = 4–5. (E) Representative H&E images showing pronounced tumor mass in the liver of control (GFP), relative to experimental (CXCL12) xenografted mice.

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: (A) Representative bioluminescence images of mice xenografted with GFP- or CXCL12-expressing cells at implantation (Day 0) or study endpoint (Day 28). (B) Whole-body in vivo radiance over time of GFP- and CXCL12-mice. (C) Ex vivo radiance of excised spleen (C), reflecting tumor cells at the site of injection, or the metastatic destination (D), reflecting decreased hepatic metastasis of CXCL12-expressing cells relative to GFP-cells. (**) denotes P ≤0.01, n = 4–5. (E) Representative H&E images showing pronounced tumor mass in the liver of control (GFP), relative to experimental (CXCL12) xenografted mice.

Techniques: Expressing, In Vivo, Ex Vivo, Injection

Apoptosis of GFP and CXCL12-expressing MiaPaCa2 cells were assessed using the caspase 3/7 glo assay.Cells were starved for 24 hours and cultured in 0% serum (A, C) or 1% serum (B, D) containing medium. (A–B) Apoptosis in adherent CXCL12-expressing cells was elevated compared to either GFP-expressing clones in serum-free conditions with no change seen in 1% serum. GFP-cells werestimulated with 100 µM gemcitabine as a control. (C–D) To measure cell number and detachment based apoptosis of cells in suspension, MiaPaCa2-luciferase cells were cultured on poly-HEMA. Using the Viacount reagent and flow cytometric cell counting, there was no difference in live cell number observed in non-adherent CXCL12-null (GFP) or expressing cells when cultured either in 0% (C) or 1% serum (D). Apoptosis of poly-HEMA cultured cells revealed an in increase in active caspase-3/7 restricted to cells cultured in serum-free conditions only (C) with no change observed in 1% serum (D). Gemcitabine (GEM) was used as a control for decreased cell count and increased apoptosis.(*), (**), and (***) denote P ≤0.05, P ≤0.01, and P ≤0.001 respectively in comparison to control cells (GFP). Values are mean±SEM, n = 4–5.

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: Apoptosis of GFP and CXCL12-expressing MiaPaCa2 cells were assessed using the caspase 3/7 glo assay.Cells were starved for 24 hours and cultured in 0% serum (A, C) or 1% serum (B, D) containing medium. (A–B) Apoptosis in adherent CXCL12-expressing cells was elevated compared to either GFP-expressing clones in serum-free conditions with no change seen in 1% serum. GFP-cells werestimulated with 100 µM gemcitabine as a control. (C–D) To measure cell number and detachment based apoptosis of cells in suspension, MiaPaCa2-luciferase cells were cultured on poly-HEMA. Using the Viacount reagent and flow cytometric cell counting, there was no difference in live cell number observed in non-adherent CXCL12-null (GFP) or expressing cells when cultured either in 0% (C) or 1% serum (D). Apoptosis of poly-HEMA cultured cells revealed an in increase in active caspase-3/7 restricted to cells cultured in serum-free conditions only (C) with no change observed in 1% serum (D). Gemcitabine (GEM) was used as a control for decreased cell count and increased apoptosis.(*), (**), and (***) denote P ≤0.05, P ≤0.01, and P ≤0.001 respectively in comparison to control cells (GFP). Values are mean±SEM, n = 4–5.

Techniques: Expressing, Glo Assay, Cell Culture, Clone Assay, Luciferase, Cell Counting

Population growth of adherent GFP and CXCL12-expressing MiaPaCa2 cells was assessed using the Viacount reagent and flow cytometric cell counting (A–B). CXCL12-expressing cells starved for 24 hours and cultured in both 0% serum (A) or 1% serum (B) containing medium were found to have decreased population growth.(B) Two CXCL12-expressing clones (#1, #2) were compared to GFP alone or GFP + gemcitabine (GEM) controls in 1% serum containing medium. Doubling time of adherent clones (T 2 ) was calculated using a linear regression of the data to determine slope and the intercept at y = 10 6 , with increased T 2 observed in both CXCL12-expressing clones. (C–D) Propidium Iodide cell cycle analysis revealed a decrease in percentage of cells in the G 2 phase in CXCL12-expressing cells compared to GFP controls. (*), (**), and (***) denote P ≤0.05, P ≤0.01, and P ≤0.001 respectively in comparison to control cells (GFP). Values are mean±SEM, n = 4–5.

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: Population growth of adherent GFP and CXCL12-expressing MiaPaCa2 cells was assessed using the Viacount reagent and flow cytometric cell counting (A–B). CXCL12-expressing cells starved for 24 hours and cultured in both 0% serum (A) or 1% serum (B) containing medium were found to have decreased population growth.(B) Two CXCL12-expressing clones (#1, #2) were compared to GFP alone or GFP + gemcitabine (GEM) controls in 1% serum containing medium. Doubling time of adherent clones (T 2 ) was calculated using a linear regression of the data to determine slope and the intercept at y = 10 6 , with increased T 2 observed in both CXCL12-expressing clones. (C–D) Propidium Iodide cell cycle analysis revealed a decrease in percentage of cells in the G 2 phase in CXCL12-expressing cells compared to GFP controls. (*), (**), and (***) denote P ≤0.05, P ≤0.01, and P ≤0.001 respectively in comparison to control cells (GFP). Values are mean±SEM, n = 4–5.

Techniques: Expressing, Cell Counting, Cell Culture, Clone Assay, Cell Cycle Assay

(A) Kaplan-Meier survival curves for GFP- and CXCL12-expressing cell groups. Three experimental CXCL12 PDAC injected mice were removed for non-study reasons (tick marks). (B) Percent change in bioluminescence from baseline-level measured at day 7 for both GFP and CXCL12 engrafted mice. Dotted lines represent individual mice. Solid lines are quadratic regression fitted curves of each group. Statistical comparison was done between both groups independent of time. (C–D) Representative bioluminescence images of mice from each groupat days 7, 49, and endpoint for GFP (98) or CXCL12 (106). (E) Tumor wet weight was significantly reduced in CXCL12-expressing tumors relative GFP-tumors. Representative photomicrographs are shown in lower panels. (F) CXCL12-producing tumors had significantly fewer Ki-67 positive cells compared to GFP-expressing tumors, as counted in a cross-section of each tumor normalized to the total cross-sectional area of each tumor. (G) Representative images of Ki-67immunostaining and rabbit isotype control (inset, Rab IgG) are shown.n = 8–10. (**) and (***) denote P ≤0.01 and P ≤0.001 respectively, between CXCL12-expressing and control xenografted mice.

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: (A) Kaplan-Meier survival curves for GFP- and CXCL12-expressing cell groups. Three experimental CXCL12 PDAC injected mice were removed for non-study reasons (tick marks). (B) Percent change in bioluminescence from baseline-level measured at day 7 for both GFP and CXCL12 engrafted mice. Dotted lines represent individual mice. Solid lines are quadratic regression fitted curves of each group. Statistical comparison was done between both groups independent of time. (C–D) Representative bioluminescence images of mice from each groupat days 7, 49, and endpoint for GFP (98) or CXCL12 (106). (E) Tumor wet weight was significantly reduced in CXCL12-expressing tumors relative GFP-tumors. Representative photomicrographs are shown in lower panels. (F) CXCL12-producing tumors had significantly fewer Ki-67 positive cells compared to GFP-expressing tumors, as counted in a cross-section of each tumor normalized to the total cross-sectional area of each tumor. (G) Representative images of Ki-67immunostaining and rabbit isotype control (inset, Rab IgG) are shown.n = 8–10. (**) and (***) denote P ≤0.01 and P ≤0.001 respectively, between CXCL12-expressing and control xenografted mice.

Techniques: Expressing, Injection

Ex vivo bioluminescence analysis revealed significantly decreased metastasis to the liver (A), lung (C), and mesenteric lymph nodes (D) of CXCL12-expressing cells compared to GFP-controls. Representative biophotonic images of hepatic metastases are shown in panel B. (**) and (***) denote statistically significant P ≤0.01 and P ≤0.001, respectively, differences between CXCL12-expressing and control tumor engrafted mice. n = 8–10 mice in each group.

Journal: PLoS ONE

Article Title: CXCL12 Chemokine Expression Suppresses Human Pancreatic Cancer Growth and Metastasis

doi: 10.1371/journal.pone.0090400

Figure Lengend Snippet: Ex vivo bioluminescence analysis revealed significantly decreased metastasis to the liver (A), lung (C), and mesenteric lymph nodes (D) of CXCL12-expressing cells compared to GFP-controls. Representative biophotonic images of hepatic metastases are shown in panel B. (**) and (***) denote statistically significant P ≤0.01 and P ≤0.001, respectively, differences between CXCL12-expressing and control tumor engrafted mice. n = 8–10 mice in each group.

Techniques: Ex Vivo, Expressing

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