anti cd34 af488  (Bioss)

Journal: EBioMedicine

Article Title: A liquid biopsy for detecting circulating mesothelial precursor cells: A new biomarker for diagnosis and prognosis in mesothelioma

doi: 10.1016/j.ebiom.2020.103031

Figure Lengend Snippet: Overview of mesothelial precursor cells (MPC) analysis using a microfluidic approach. (a) Sample preparation for MPC analysis is quick and simple. MSLN+ cells are tagged with biotinylated anti-MSLN antibody and further conjugated with anti-biotin magnetic nanoparticle (MNP) complex. Magnetically labeled samples are loaded into the microfluidic chip at 500µL/h with external magnets placed above and below the device to facilitate magnetic trapping of targeted cells. (b) Multiple populations of MPC exist in circulation and are subcategorized based on varying combinations of CD34 and/or CD90 in conjunction with mesothelin (MSLN). Image created with BioRender. (c) Overview of MesoFind microfluidic chip for MPC isolation. The microfluidic device contains 8 different zones with a sequential increase in height from 50µm to 400µm (inlet-to-outlet) and features varying average linear flow velocities (1.0x, 0.50x, 0.33x, 0.25x, 0.20x, 0.17x, 0.14x, 0.13x). Cells magnetically labeled with mesothelin are captured in different zones based on the relative amount of magnetic content. Cells with higher MSLN expression (high magnetic content) are captured in earlier zones whereas cells with less MSLN (low magnetic content) are captured in later zones. Captured cells are fluorescently labeled with CD34 and CD90 antibodies to identify and isolate different MPC subpopulations. (d) MSLN expression profiles are generated for each MPC subpopulation based on the captured location on the chip. Each MPC subtypes are predicted to exhibit different clinical characteristics in patients and their expression of MSLN correlates to the severity of the conditions.

Article Snippet: The captured cells were then immunostained with 200µL of an antibody cocktail containing either 2mg/mL of anti-CD34 AF488 (bs-8996R-A488, Bioss Antibodies, USA), 2mg/mL of anti-CD90 AF555 (bs-0778R-A555, Bioss Antibodies, USA), and 0.6 mg/mL anti-CD45 APC (17-0451-83, Invitrogen, USA) for mice-related samples or 1mg/mL of anti-CD34 AF488 (ab195013, Abcam, Canada), 0.5mg/mL of anti-CD90 AF555 (bs-10430R-A555, Bioss Antibodies, USA), and 1.2% v/v of anti-CD45 APC (555485, BD Biosciences, USA) for human-related samples.

Techniques: Sample Prep, Labeling, Isolation, Expressing, Generated

Journal: EBioMedicine

Article Title: A liquid biopsy for detecting circulating mesothelial precursor cells: A new biomarker for diagnosis and prognosis in mesothelioma

doi: 10.1016/j.ebiom.2020.103031

Figure Lengend Snippet: Validation of the microfluidic approach for MPC analysis. (a) Captured efficiency of mice mesothelioma cell line (RN5) and human mesothelioma cell lines (H2052 and H2452) spiked in healthy blood using MesoFind microfluidic chips. (b) Identification of different MPC and leukocytes from blood using fluorescence microscopy after cell entrapment using MesoFind devices. Three differing MPC subtypes (top three) shown here contains different combinations of CD34 and/or CD90 expression and were negative for CD45. Leukocytes shown express CD34- CD90- CD45+ but were generally identified as CD45+ and can have varying expressions of CD34 and CD90 (not shown). All scale bars are 20 um. (c) Schematic of mesothelioma mice model analysis. RN5 mesothelioma cells were injected intraperitoneally (i.p.) and mice were sacrificed between 1 to 6 weeks post-injection. Control mice were injected with saline. The lavage and blood collected from the mice were analyzed using flow cytometry or MesoFind microfluidic analysis followed by fluorescence microscopy for the identification of different MPC populations. Images were created with BioRender. (d) Total MSLN+ CD34+ CD90+ MPC captured from the lavage collected from mice using MesoFind analysis. A general increase in total counts observed from 0 to 6 weeks post-injection. (e) Flow cytometry of MSLN+ CD34+ CD90+ MPC in lavage samples. No significant change was observed after 4-weeks post-injection. No MPC were detected in blood samples using flow cytometric analysis (not shown). (f) Total MSLN+ CD34+ CD90+ MPC captured from the blood of mice using MesoFind analysis. A general increase in total counts observed from 0 to 6 weeks post-injection. (g) Mesothelin expression indices (EI) for MSLN+ CD34+ CD90+ MPC in lavage and blood samples from 0 to 6 weeks post-injection using MesoFind analysis. (h) Flow cytometric gating for MSLN+ cells and CD34+ CD90+ cells in naive mice and (i) 4 weeks RN5 post-injected mice. All analysis was performed in triplicates (n=3). Error bars for Fig. 2 d,f, and g represent standard error of mean. All reported values were compared to values at 0 week (d,e,f) or naïve mice (g) using unpaired t-test, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The captured cells were then immunostained with 200µL of an antibody cocktail containing either 2mg/mL of anti-CD34 AF488 (bs-8996R-A488, Bioss Antibodies, USA), 2mg/mL of anti-CD90 AF555 (bs-0778R-A555, Bioss Antibodies, USA), and 0.6 mg/mL anti-CD45 APC (17-0451-83, Invitrogen, USA) for mice-related samples or 1mg/mL of anti-CD34 AF488 (ab195013, Abcam, Canada), 0.5mg/mL of anti-CD90 AF555 (bs-10430R-A555, Bioss Antibodies, USA), and 1.2% v/v of anti-CD45 APC (555485, BD Biosciences, USA) for human-related samples.

Techniques: Fluorescence, Microscopy, Expressing, Injection, Flow Cytometry

Journal: EBioMedicine

Article Title: A liquid biopsy for detecting circulating mesothelial precursor cells: A new biomarker for diagnosis and prognosis in mesothelioma

doi: 10.1016/j.ebiom.2020.103031

Figure Lengend Snippet: Clinical utility of analyzing various MPC subpopulations using MesoFind device. (a) Total blood counts for MSLN+ CD34+ CD90+ MPC from healthy donor (HD, n=10), asbestos-exposed individuals (ASB, n=23), and malignant pleural mesothelioma patients (MPM, n=23) using MesoFind devices. (b) Comparison of mesothelin expression indices between varying subpopulations of MPC (CD90+ CD34-, CD90- CD34+, CD90+ CD34+, CD90+, CD34+) in HD, ASB, and MPM individuals. All MPC were MSLN+ and CD45-. (c-h) MSLN expression indices (EI) for most representative MPC subtypes in each clinical evaluation. Overall, higher MSLN expression indices corresponded to higher severity in clinical tests. EI cut-off values between each group were determined and the clinical sensitivity and specificity were reported (right). (c) EI of MSLN+ CD90- CD34+ CD45- MPC in ASB (n=23) and MPM (n=23). Cut-off EI=500. (d) EI of MSLN+ CD90+ CD34- CD45- MPC in HD (n=10) and ASB (n=23). Cut-off EI=200. (e) EI of MSLN+ CD90+ CD45- MPC were compared between epithelioid (n=16) and biphasic (n=5) cancer subtypes. Cut-off EI = 2650. (f) EI of MSLN+ CD34+ CD45- MPC in patients with low (<6) (n=4) and high (>6) (n=4) SUV scores through PET scans were compared. Cut-off EI=6700. (g) EI of MSLN+ CD90+ CD34- CD45- MPC for patients with unresectable tumors (n=19) and resectable tumors (n=4) were compared. Cut-off EI=200. (h) Presence (n=3) and absence (n=13) of lymph node metastasis was compared through MSLN+ CD34+ CD45- MPC. Cut-off EI=24500. Statistics are performed with unpaired t-tests, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The captured cells were then immunostained with 200µL of an antibody cocktail containing either 2mg/mL of anti-CD34 AF488 (bs-8996R-A488, Bioss Antibodies, USA), 2mg/mL of anti-CD90 AF555 (bs-0778R-A555, Bioss Antibodies, USA), and 0.6 mg/mL anti-CD45 APC (17-0451-83, Invitrogen, USA) for mice-related samples or 1mg/mL of anti-CD34 AF488 (ab195013, Abcam, Canada), 0.5mg/mL of anti-CD90 AF555 (bs-10430R-A555, Bioss Antibodies, USA), and 1.2% v/v of anti-CD45 APC (555485, BD Biosciences, USA) for human-related samples.

Techniques: Expressing

Journal: EBioMedicine

Article Title: A liquid biopsy for detecting circulating mesothelial precursor cells: A new biomarker for diagnosis and prognosis in mesothelioma

doi: 10.1016/j.ebiom.2020.103031

Figure Lengend Snippet: Overview of mesothelial precursor cells (MPC) analysis using a microfluidic approach. (a) Sample preparation for MPC analysis is quick and simple. MSLN+ cells are tagged with biotinylated anti-MSLN antibody and further conjugated with anti-biotin magnetic nanoparticle (MNP) complex. Magnetically labeled samples are loaded into the microfluidic chip at 500µL/h with external magnets placed above and below the device to facilitate magnetic trapping of targeted cells. (b) Multiple populations of MPC exist in circulation and are subcategorized based on varying combinations of CD34 and/or CD90 in conjunction with mesothelin (MSLN). Image created with BioRender. (c) Overview of MesoFind microfluidic chip for MPC isolation. The microfluidic device contains 8 different zones with a sequential increase in height from 50µm to 400µm (inlet-to-outlet) and features varying average linear flow velocities (1.0x, 0.50x, 0.33x, 0.25x, 0.20x, 0.17x, 0.14x, 0.13x). Cells magnetically labeled with mesothelin are captured in different zones based on the relative amount of magnetic content. Cells with higher MSLN expression (high magnetic content) are captured in earlier zones whereas cells with less MSLN (low magnetic content) are captured in later zones. Captured cells are fluorescently labeled with CD34 and CD90 antibodies to identify and isolate different MPC subpopulations. (d) MSLN expression profiles are generated for each MPC subpopulation based on the captured location on the chip. Each MPC subtypes are predicted to exhibit different clinical characteristics in patients and their expression of MSLN correlates to the severity of the conditions.

Article Snippet: The captured cells were then immunostained with 200µL of an antibody cocktail containing either 2mg/mL of anti-CD34 AF488 (bs-8996R-A488, Bioss Antibodies, USA), 2mg/mL of anti-CD90 AF555 (bs-0778R-A555, Bioss Antibodies, USA), and 0.6 mg/mL anti-CD45 APC (17-0451-83, Invitrogen, USA) for mice-related samples or 1mg/mL of anti-CD34 AF488 (ab195013, Abcam, Canada), 0.5mg/mL of anti-CD90 AF555 (bs-10430R-A555, Bioss Antibodies, USA), and 1.2% v/v of anti-CD45 APC (555485, BD Biosciences, USA) for human-related samples.

Techniques: Sample Prep, Labeling, Isolation, Expressing, Generated

Journal: EBioMedicine

Article Title: A liquid biopsy for detecting circulating mesothelial precursor cells: A new biomarker for diagnosis and prognosis in mesothelioma

doi: 10.1016/j.ebiom.2020.103031

Figure Lengend Snippet: Validation of the microfluidic approach for MPC analysis. (a) Captured efficiency of mice mesothelioma cell line (RN5) and human mesothelioma cell lines (H2052 and H2452) spiked in healthy blood using MesoFind microfluidic chips. (b) Identification of different MPC and leukocytes from blood using fluorescence microscopy after cell entrapment using MesoFind devices. Three differing MPC subtypes (top three) shown here contains different combinations of CD34 and/or CD90 expression and were negative for CD45. Leukocytes shown express CD34- CD90- CD45+ but were generally identified as CD45+ and can have varying expressions of CD34 and CD90 (not shown). All scale bars are 20 um. (c) Schematic of mesothelioma mice model analysis. RN5 mesothelioma cells were injected intraperitoneally (i.p.) and mice were sacrificed between 1 to 6 weeks post-injection. Control mice were injected with saline. The lavage and blood collected from the mice were analyzed using flow cytometry or MesoFind microfluidic analysis followed by fluorescence microscopy for the identification of different MPC populations. Images were created with BioRender. (d) Total MSLN+ CD34+ CD90+ MPC captured from the lavage collected from mice using MesoFind analysis. A general increase in total counts observed from 0 to 6 weeks post-injection. (e) Flow cytometry of MSLN+ CD34+ CD90+ MPC in lavage samples. No significant change was observed after 4-weeks post-injection. No MPC were detected in blood samples using flow cytometric analysis (not shown). (f) Total MSLN+ CD34+ CD90+ MPC captured from the blood of mice using MesoFind analysis. A general increase in total counts observed from 0 to 6 weeks post-injection. (g) Mesothelin expression indices (EI) for MSLN+ CD34+ CD90+ MPC in lavage and blood samples from 0 to 6 weeks post-injection using MesoFind analysis. (h) Flow cytometric gating for MSLN+ cells and CD34+ CD90+ cells in naive mice and (i) 4 weeks RN5 post-injected mice. All analysis was performed in triplicates (n=3). Error bars for Fig. 2 d,f, and g represent standard error of mean. All reported values were compared to values at 0 week (d,e,f) or naïve mice (g) using unpaired t-test, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The captured cells were then immunostained with 200µL of an antibody cocktail containing either 2mg/mL of anti-CD34 AF488 (bs-8996R-A488, Bioss Antibodies, USA), 2mg/mL of anti-CD90 AF555 (bs-0778R-A555, Bioss Antibodies, USA), and 0.6 mg/mL anti-CD45 APC (17-0451-83, Invitrogen, USA) for mice-related samples or 1mg/mL of anti-CD34 AF488 (ab195013, Abcam, Canada), 0.5mg/mL of anti-CD90 AF555 (bs-10430R-A555, Bioss Antibodies, USA), and 1.2% v/v of anti-CD45 APC (555485, BD Biosciences, USA) for human-related samples.

Techniques: Fluorescence, Microscopy, Expressing, Injection, Flow Cytometry

Journal: EBioMedicine

Article Title: A liquid biopsy for detecting circulating mesothelial precursor cells: A new biomarker for diagnosis and prognosis in mesothelioma

doi: 10.1016/j.ebiom.2020.103031

Figure Lengend Snippet: Clinical utility of analyzing various MPC subpopulations using MesoFind device. (a) Total blood counts for MSLN+ CD34+ CD90+ MPC from healthy donor (HD, n=10), asbestos-exposed individuals (ASB, n=23), and malignant pleural mesothelioma patients (MPM, n=23) using MesoFind devices. (b) Comparison of mesothelin expression indices between varying subpopulations of MPC (CD90+ CD34-, CD90- CD34+, CD90+ CD34+, CD90+, CD34+) in HD, ASB, and MPM individuals. All MPC were MSLN+ and CD45-. (c-h) MSLN expression indices (EI) for most representative MPC subtypes in each clinical evaluation. Overall, higher MSLN expression indices corresponded to higher severity in clinical tests. EI cut-off values between each group were determined and the clinical sensitivity and specificity were reported (right). (c) EI of MSLN+ CD90- CD34+ CD45- MPC in ASB (n=23) and MPM (n=23). Cut-off EI=500. (d) EI of MSLN+ CD90+ CD34- CD45- MPC in HD (n=10) and ASB (n=23). Cut-off EI=200. (e) EI of MSLN+ CD90+ CD45- MPC were compared between epithelioid (n=16) and biphasic (n=5) cancer subtypes. Cut-off EI = 2650. (f) EI of MSLN+ CD34+ CD45- MPC in patients with low (<6) (n=4) and high (>6) (n=4) SUV scores through PET scans were compared. Cut-off EI=6700. (g) EI of MSLN+ CD90+ CD34- CD45- MPC for patients with unresectable tumors (n=19) and resectable tumors (n=4) were compared. Cut-off EI=200. (h) Presence (n=3) and absence (n=13) of lymph node metastasis was compared through MSLN+ CD34+ CD45- MPC. Cut-off EI=24500. Statistics are performed with unpaired t-tests, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The captured cells were then immunostained with 200µL of an antibody cocktail containing either 2mg/mL of anti-CD34 AF488 (bs-8996R-A488, Bioss Antibodies, USA), 2mg/mL of anti-CD90 AF555 (bs-0778R-A555, Bioss Antibodies, USA), and 0.6 mg/mL anti-CD45 APC (17-0451-83, Invitrogen, USA) for mice-related samples or 1mg/mL of anti-CD34 AF488 (ab195013, Abcam, Canada), 0.5mg/mL of anti-CD90 AF555 (bs-10430R-A555, Bioss Antibodies, USA), and 1.2% v/v of anti-CD45 APC (555485, BD Biosciences, USA) for human-related samples.

Techniques: Expressing