Normal human bone marrow aspirates were obtained with written consent from healthy donors in accordance with the Declaration of Helsinki and with the approval of the Human subjects Ethics Committee of Second Affiliated Hospital of Zhejiang University. MSCs from 3 such donors were cultured as described previously [10 (link),12 (link),19 (link),74 (link)]. Cell surface markers were profiled using a BD FACS CantoTM II Flow Cytometry System after 3–5 passages as described previously [75 (link)] with the following human specific monoclonal antibodies: CD29-phycoerythrin (PE) (eBioscience, San Diego, CA, USA), CD34-PE (MACS, Miltenyi Biotec, Auburn, CA, USA) and CD166-PE (BD Biosciences Pharmingen, San Diego, CA, USA), respectively.
Facscantotmii flow cytometry system
Manufactured by BD
Sourced in United States, Germany
The BD FACSCantoTMII Flow Cytometry System is a compact, multi-color flow cytometry platform that enables the analysis and sorting of cells and particles. It provides high-performance and flexibility for a variety of applications in research and clinical settings.
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7 protocols using facscantotmii flow cytometry system
1
Isolation and Characterization of Human Mesenchymal Stem Cells
2
Quantification of Platelet Activation Markers
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For the measurement of P-selectin (CD62P) expression, platelets (from 5 μL PRP or washed platelets) were labeled after incubation with the agonists using APC-labeled mouse anti-human-CD61 antibody (Millipore), PE-labeled mouse anti-human-CD62P antibody (BD Pharmingen), and the appropriate isotype control (mouse IgG1 PE, Santa Cruz Biotechnology). Platelet suspensions were added to the antibody dilutions (1:20) and incubated for 20 min at RT, protected from light. Samples were then measured using a BD FACSCantoTM II Flow Cytometry System (BD Biosciences) (Figure S7 ).
For platelet aggregation, 0.1 µg/mL PGI2 were added immediately after blood collection, and the blood was incubated for 10 min at RT. Then, the blood was centrifuged for 15 min at 150× g without a brake, and the supernatant was carefully removed without disturbing the milky interface layer, generating PRP. For background calibration, one aliquot of PRP was centrifuged at 10,000× g to pellet residual platelets and gain PPP. Aggregation measurements were performed on an APACT 4004 aggregometer, pre-heated to 37 °C. Stirring was set to 1000 rpm. One hundred and fifty-six microliters of PRP were used for each measurement, and 4 µL of supplement/PBS control were added before starting the data collection (160 µL total volume). Light transmission at 740 nm was determined, and data were collected for 420 s (Figure S7 ).
For platelet aggregation, 0.1 µg/mL PGI2 were added immediately after blood collection, and the blood was incubated for 10 min at RT. Then, the blood was centrifuged for 15 min at 150× g without a brake, and the supernatant was carefully removed without disturbing the milky interface layer, generating PRP. For background calibration, one aliquot of PRP was centrifuged at 10,000× g to pellet residual platelets and gain PPP. Aggregation measurements were performed on an APACT 4004 aggregometer, pre-heated to 37 °C. Stirring was set to 1000 rpm. One hundred and fifty-six microliters of PRP were used for each measurement, and 4 µL of supplement/PBS control were added before starting the data collection (160 µL total volume). Light transmission at 740 nm was determined, and data were collected for 420 s (
3
BD FACSCanto II Flow Cytometry Analysis
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BD FACSCantoTMII Flow Cytometry System with BD FACSDIVATM Software
6.1.3 software (BD Biosciences Heidelberg, Germany) and scil Vet abc™ Hematology Analyzer
(scil animal care co. GmbH, Viernheim, Germany) were used to acquire the data. Limit
values stated in the following comply with scil Vet standards. Graphs were created using
GraphPad Prism 5 (v5.02, GraphPad Software Inc., La Jolla, CA, USA). The results are
represented as mean ± SD. Data was checked for Gaussian distribution by D’Agostino and
Pearson omnibus normality test using GraphPad Prism. P values were
determined by analysis of variances (ANOVA) for data that passed the normality test
(α=0.05) or by Kruskal-Wallis test for non-normally distributed data using GraphPad Prism.
Log-Rank test and pair-wise multiple comparison of means (Holm-Sidak method) were used for
the analysis of Kaplan-Meier survival curves. A P value <0.05
indicates significant differences Asterisks in figures are used as follows:
*P<0.05, **P<0.01,
***P<0.001, ****P<0.0001.
6.1.3 software (BD Biosciences Heidelberg, Germany) and scil Vet abc™ Hematology Analyzer
(scil animal care co. GmbH, Viernheim, Germany) were used to acquire the data. Limit
values stated in the following comply with scil Vet standards. Graphs were created using
GraphPad Prism 5 (v5.02, GraphPad Software Inc., La Jolla, CA, USA). The results are
represented as mean ± SD. Data was checked for Gaussian distribution by D’Agostino and
Pearson omnibus normality test using GraphPad Prism. P values were
determined by analysis of variances (ANOVA) for data that passed the normality test
(α=0.05) or by Kruskal-Wallis test for non-normally distributed data using GraphPad Prism.
Log-Rank test and pair-wise multiple comparison of means (Holm-Sidak method) were used for
the analysis of Kaplan-Meier survival curves. A P value <0.05
indicates significant differences Asterisks in figures are used as follows:
*P<0.05, **P<0.01,
***P<0.001, ****P<0.0001.
4
Thioflavin-S Assay for Tau Expression in N2aTau4RDΔK280 Cells
5
Tau Aggregation Inhibition Screening
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Cell suspensions of inducible N2aTau4RDΔK280 cells were distributed on 24-well pates (1 ml per well) and 0.0005% (12 µM) Thioflavin-S was added to each sample. For induction of Tau expression 1 µg/ml doxycyclin was added, except for the uninduced negative control. 60 µM compounds were added, except for the untreated positive control (all compounds were tested at 60 µM concentration). An additional assay was performed with some selected compounds in the concentration range of 0 to 60 µM (Fig. 6B-D ; Table 4 ). The cells were incubated for 4 days at 37 °C. Floating and adherent cells were combined, pelleted (5 min, 295 g) and washed once with PBS. Cells were then counted in a BD FACSCantoTMII Flow Cytometry System by the fluorescent signal intensity in the FITC channel (Excitation: 495 nm, Emission: 519 nm).
6
Berberine Attenuates AAPH-Induced Apoptosis
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C17.2 cells (1 × 106/well) were cultured in 6-well plate in proliferation medium for 24 h. AAPH (7.38 mM) and berberine (1.69 mM) was added to treat C17.2 cell for 12 h, while only AAPH (7.38) added was set as control. After 12-h treatment, cells were collected and stained with FITC Annexin V and propidium iodide (PI) in the dark using a FITC Annexin V and PI Apoptosis Kit (A026, GeneCopoeia), according to the manufacturers’ instruction. After being washed, the apoptotic cells were determined by a BD FACSCantoTM II Flow Cytometry System.
7
Isolation and Stimulation of CD14+ PBMCs
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