Cd49d apc

The CLL cells from all the blood samples were stained with optimal concentrations of antibody combinations and directed against the following surface antigens: CD183(CXCR3)-FITC, CD20-PE, CD5-PerCP-Cy5.5, CD38-Pe-Cy7, CD49d-APC, CD19-APC-Cy7, CD184 (CXCR4)-BV421 and HLA-DR-BV510 (all procured from BioLegend), as previously reported [5 (link),15 (link)]. Isotype-matched antibodies (BioLegend) were used as negative controls.
The determination of s-CLL and l-CLL cells was conducted using FSC data and a back-gating strategy. The analysis was performed using a BD FACSCanto II (Becton Dickinson) instrument, and data acquisition was performed using BD FACSDiva software (v.8.0.2; Becton Dickinson). Flow cytometry data were analysed using FlowJo v.X0.7 software (Tree Star, Inc., San Carlos, CA, USA). In all the experiments, a minimum of 10,000 events was counted. The results were expressed as a percentage and mean fluorescence intensity (MFI).

For primary monocytes, 20 µl of citrate blood was diluted in 80 µl PBS for staining within 4 h after blood collection. Primary monocytes and THP-1 cells were stained with following antibodies (diluted 1:50): CD45-PE (Cat: 368510), CD14-FITC (Cat: 367116), CD16-BV-510 (Cat: 302048), HLA-DR-APC-Cy7 (Cat: 307618), CX3CR1-BV421 (Cat: 341620), CD11a-PE-Cy7 (Cat: 301220), CD11b-PerCP (Cat: 101230), CD11c-BV421 (Cat: 371512), CD29-PE-CY7 (Cat: 303026), and CD49d-APC (Cat: 304308) (all from Biolegend, San Diego, USA). After 25 min staining in the dark, 650 µl RBC Lysis Buffer (Biolegend) were added to the samples and incubated for another 20 min. Subsequently, suspension was centrifuged at 400 × g for 5 min and supernatant was discarded. Cell pellet was resuspended in 100 µl fresh PBS and used for FACS analysis (Supplementary Figure 1S). Flow cytometry was performed with a MACSQuant 10 flow cytometer (Miltenyi Biotec, Bergisch-Gladbach, Germany) and data were analyzed using the FlowJo software version 10.0 (FlowJo, LLC, Ashland, USA).

The delivery efficiency of ASO or Toc-HDO into primary T cells in vivo and EL4 in vitro cells was assayed by flow cytometry using Alexa Flour 647-labeled ASO. Cells were harvested at the indicated time points and washed in PBS by centrifugation at 500 ×;g for 5 min. The cells were then washed and suspended in PBS with 2% BSA and 0.05% sodium azide (Sigma-Aldrich). Surface marker expression levels were determined using the following antibodies: CD3-PE (BioLegend, clone 17A2, 100205), CD45R/B220-PECy7 (BioLegend, clone RA3-6B2, #103221) or CD45R/B220-FITC (BioLegend, clone RA3-6B2, #103205), and CD49d-APC (BioLegend, clone R1-2, #103621). All related antibodies are listed in Supplementary Table 2. Cells were then sorted using a BD FACSAria cell sorter, BD FACSDiva software, and FlowJo v10 software (BD Biosciences) or analyzed using a BD FACSVerse cell analyzer and BD FACSuite software (BD Biosciences). The sorting/gating strategy is shown in Supplementary Fig. 2g.