Cd5 pc5

Platelets were gated using CD42b-APC and CD61-PE (#551061, #IM3605; Beckman Coulter) antibodies. For the isolation of leukocytes, whole blood was drawn into Vacutainer EDTA tubes. Erythrocytes were lysed using a TQ-Prep Workstation (Beckman Coulter) and the remaining cells were washed with PBS. Lymphocytes and monocytes were gated using CD5-PC5.5 and CD14-PC7 antibodies (#B49191, #A22331; Beckman Coulter), respectively. After exclusion of lymphocytes and monocytes, granulocytes were gated by forward (FSC) and side scatter (SSC) characteristics. α-2,6- and α-2,3-linked sialic acid was detected using 250 ng/mL Cy5-labeled Sambucus nigra lectin (SNA) (#CL-1305; Vector Laboratories) and 100 μg/mL FITC-labeled Maackia amurensis lectin II (MAL II) (#21511103-1; GlycoMatrix), respectively. Cells were acquired on a CytoFLEX flow cytometer (Beckman Coulter) and analyzed with FlowJo software (Tree Star). To adjust for distinct autofluorescent properties of investigated cells, results were presented as delta geometric mean fluorescence intensity (Δ gMFI) in relation to buffer treated cells.

Intracellular staining of Ki-67 was performed using a fluorescein isothiocyanate (FITC)-labeled antibody against Ki-67 (Becton Dickinson, Franklin Lakes, NJ, USA) after fixation and permeabilization using the BD Intrasure kit (Becton Dickinson) following the manufacturer's instructions. Surface staining of cells was performed using the following monoclonal antibodies conjugated with the indicated fluorochrome: CD19-phycoerythrin (PE) and CD5-allophycocyanine (APC) (Becton Dickinson). To characterize the phenotype of proliferative and resting compartments of CLL cells,we used the following antibodies: CD19-energy coupled dye (ECD), CD5-phycoerythrincyanine 5.5 (PC5.5) (Beckman Coulter, Brea, CA, USA), CD3-PE-cyanine 7 (Cy7), CXCR4-APC, CXCR5-APC, CCR7-APC, CD49d-APC, CD62L-APC, Ki-67-FITC (Becton Dickinson), and CD38-PE (EBioscience, San Diego, CA, USA). The rates of T cell activation and proliferation were analyzed by determining the expression of Ki-67, CD69 and CD38 in CD3+ cellsusing the following antibodies: Ki-67-FITC, CD38-PE (EBioscience), CD5-PC5.5 (Beckman Coulter), CD3-PE-Cy7 and CD69-APC (Becton Dickinson). Cells were acquired in a Navios^TM cytometer (Beckman Coulter) and the results were analyzed using the FCS Express 4 software (De Novo Software, Los Angeles, CA, USA).

3D bioprinted scaffolds were smashed with 500µl of dissolution buffer (26 (link)), passed through a 30µm CellTrics filter (Sysmex, Kobe, Japan) to flow cytometer tubes, and eventually stained for 25 minutes RT for the following antibodies: CD5 PC5 (Beckman Coulter, Brea, USA), CD19 PC7 (Beckman Coulter, Brea, USA), and IgM PE (Miltenyi, Bergisch Gladbach, Germany). After washing with PBS 1500RPM, 5 minutes, cells were analyzed on Navios Flow Cytometer (Beckman Coulter, Brea, USA). Analyses were performed with the FCS Express software (DeNovo Software). Representative density plots were normalized on equal numbers of events occurring in the gates of interest. 3D bioprinted scaffold without cells was stained with the antibodies we used, in order to exclude nonspecific binding.