Anti hla dr apc

The isolated CD16⁻ and CD16⁺ monocytes were double-stained with anti-CD16-PE (BD Biosciences, USA) and anti-TLR4-APC (BD Biosciences, USA) antibodies. In a dose-finding preliminary experiment, the most potent stimulation of TLR4 expression on cells analyzed by flow cytometry (FACScan, BD Biosciences) was presented at 100ng/mL of Escherichia Coli LPS (Sigma-Aldrich, St, Louis, MO). Therefore, all the following acute in vitro experiments were divided into un-stimulated and stimulated [LPS, 100ng/mL] groups. The HLA-DR expression on CD16^- (classical) and CD16⁺ (non-classical) monocytes were measured by staining with anti-HLA-DR-APC, anti-CD14-FITC (BD Biosciences, USA) and anti-CD16-PE antibodies.
Our preliminary experiments revealed that the TLR4 expression was not different between CD16^- (classical) monocytes between groups. It is well-established that classical monocyte are phagocytic with no inflammatory attributes [15 (link)–17 (link)], so the LPS-stimulated extra- and intra-cellular inflammatory cytokines production and corresponding signals were only evaluated in cultured CD16⁺ (non-classical) monocytes.

Antibodies used for surface staining included anti-CD14 Qdot 655, anti-CD20 Qdot 655, anti-CD4 PeCy5.5, anti-CD8 Qdot605 (Invitrogen; Carlsbad, California), anti-CD14 BV650, anti-CD20 BV650 (Biolegend; San Diego, California), anti-CD28 ECD (Beckman Coulter; Fullerton, CA), anti-CD25 APC-Cy7, anti-CD95 PE-Cy5, and anti-HLA-DR APC (BD Pharmingen; San Diego, California). Antibodies used for intracellular staining included anti-interleukin-2 (IL-2) Alexa700 (Biolegend), anti-interferon-gamma (IFNγ) Alexa 700 (Invitrogen), anti-tumor necrosis factor-alpha (TNFα) Alexa 700, anti-CD3 Pac Blue, anti-CD69 PE, and anti-Ki67 FITC (BD Pharmingen).

The following flow cytometry antibodies were purchased from BD Biosciences (San Jose, CA): anti-CD3-PE Cy7, anti-CD4-Alexa488, anti-CD8-PacBlu, anti-CD28-PE, anti-CD57-APC, anti-CD45RO-Alexa 700, anti-HLA-DR-APC, anti-CD14-PacBlu, anti-CD11a-FITC, anti-CD16-PE-Cy7, anti-CD163-PE, anti-CD62L-APC, and anti-CD86-Alexa 700. Two multi-color panels were used for T cells, and two panels were used for monocytes. Data were collected using a BD LSRII flow cytometer and analyzed with FCS Express software (DeNovo Software, Los Angeles, CA).

5-Azacytidine was obtained from Sigma-Aldrich (Munich, Germany) and used at a final concentration of 5 μM or 20 μM. All flow cytometry experiments were performed using a FACS Canto II (BD Bioscience, Heidelberg, Germany). For flow cytometry the following antibodies were used: anti-CD3-PE, anti-CD4-FITC, anti-CD8-APC, anti-CD25-PE, anti-CD45-FITC, anti-CD45RO-PE, anti-CD45RA-FITC, anti-CD62L-PE, anti-CCR7-PE, anti-HLA-DR-APC, anti-Granzyme-PE antibodies (obtained from BD Bioscience) anti-CD127-PC5, anti-IFNγ-PE/Cy7, anti-IL17-APC, anti-IL4-FITC, and anti-FoxP3-APC antibodies obtained from eBioscience (San Diego, USA).

The following mAbs were used for flow cytometry: anti-CD3 PerCP, anti-CD4 FITC, anti-CD4 PerCP, anti-CD4 PE-Cy7, anti-CD8 PErCP, anti-CD8 APC-Cy7, anti-CD28 FITC, anti-CD95 APC, anti-CD195 (anti-CCR5) PE, anti-Ki67 PE, anti-HLA-DR APC, and anti-HLA-DR FITC (BD Biosciences). Additionally, annexin V PE was purchased from BD Biosciences, anti-CD8 APC and anti-CD8 PE were obtained from Beckman Coulter (CA, USA), and anti-CD38 FITC was purchased from StemCell Technologies (Vancouver, Canada). Flow cytometry data were acquired on a FACSAria II (BD Biosciences). All data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

In our study, classical monocyte was marked by CD14+ CD16− HLA-DR+ referred to previous study [15 (link)]. Peripheral blood mononuclear cells were isolated from freshly obtained EDTA-anti-coagulated whole blood samples according to Ficoll PBMC isolation protocols [23 (link)], and then stained with following murine anti-human monoclonal antibodies: 5 μl of anti-CD16 PE (Clone: B73.1, cat No.: 561313), 10 μl of anti-CD14 FITC (Clone: M5E2, cat No.: 555397), and 10 μl of anti-HLA-DR APC (Clone: TU36, cat No.: 559868) (all from BD Biosciences) and incubated for 30 min at room temperature in the dark. Next, cells were washed twice with cold phosphate-buffered saline and fixed with Cell Fix (BD Biosciences) [24 (link)]. Flow cytometry was performed using FC500 MPL software (Beckman Coulter) and data of CMF were analyzed using MXP Cytometer software (version 2.0, Beckman Coulter) (Supplementary Figure S1A,B).

During the passaging of hUC-MSCs, their morphology was observed by an inverted microscope (CKX41, OLYMPUS, Tokyo, Japan).
To test the multilineage differentiation capacity of the hUC-MSCs, adipogenic, chondrogenic, and osteogenic differentiation kits (VivaCell, Shanghai, China) were used following the manufacturer’s specifications. Finally, adipogenic, chondrogenic, and osteogenic differentiation results were tested through Oil red O staining, Alcian blue staining, and Alizarin red staining, respectively.
Surface markers on hUC-MSCs were evaluated by flow cytometry (Canto II, BD Biosciences, CA, USA). Briefly, hUC-MSCs were washed twice with staining buffer (BD Biosciences) and stained with anti-CD11b-PE, anti-CD19-FITC, anti-CD34-PE, anti-CD45-APC-Cy7, anti-CD73-PE, anti-CD90-FITC, anti-CD105-APC, anti-HLA-DR-APC, and the corresponding isotype control antibodies (all antibodies were purchased from BD Biosciences) for 30 min at room temperature in the dark. Then, the hUC-MSCs were washed twice with staining buffer and resuspended in staining buffer for flow cytometry analysis.