Cd14 macs beads

Peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation using Lymphoset, Lymphozyte Separation Media (Biowest, France), density 1.077 g/ml. After that, monocytes from the PBMC fraction were obtained by FACS. Cells were resuspended in 150 μl of staining buffer (Cell Staining Buffer, Sony, Japan). Monocytes were defined as CD45+CD56-CD14+7-AAD- population. Conjugated monoclonal antibodies to CD45, CD56, CD14, and 7-AAD were added to the cell suspension (online Table S4). Samples were analyzed on a MoFlo XDP cell sorter (Beckman Coulter, USA). Sorting of monocytes was carried out in the Purify 1–2 mode, the sorting efficiency was 70%, and the purity of the target population was 96%–99% (Figure S2). Monocytes for real-time PCR analysis were isolated from peripheral blood by density gradients followed by positive magnetic selection using CD14+ MACS beads (no. 130-050-201, Miltenyi Biotec, Germany), resulting in 90%–98% monocyte purity as confirmed by flow cytometry.

PBMCs were isolated with Lymphoprep (GE Healthcare) and CD14 MACS beads (Miltenyi) according to manufacturer's instructions.

Monocyte isolation was performed with FACS and CD14+ magnetic separation. For RNA sequencing, the peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation using Lymphosep, Lymphocyte Separation Media (#L0560-500, Biowest, France), density 1.077 g/ml. After that, monocytes from PBMC fraction were obtained by FACS. Cells were resuspended in 150 μl of staining buffer (#2701005, Cell Staining Buffer, Sony, Japan). Monocytes were defined as CD45+CD56-CD14+7-AAD- population. Conjugated monoclonal antibodies to CD45, CD56, CD14, 7-AAD were added to the cell suspension. Samples were analyzed on a MoFlo XDP cell sorter (RRID : SCR_019665, Beckman Coulter, USA). Sorting of monocytes was carried out in the Purify 1-2 mode, the sorting efficiency was 70%, the purity of the target population was 96-99%.
Monocytes for real-time PCR were isolated from peripheral blood by density gradients followed by positive magnetic selection using CD14+ MACS beads (#130-050-201, Miltenyi Biotech, Germany), resulting to 90–98% monocyte purity as confirmed by flow cytometry.
Monocytes were obtained from the following experimental points: primary before treatment (1 (link)), after NAC (2 (link)), and after surgery (3 (link)) for patients with rectal cancer; and before surgery (1 (link)) and after surgery (2 (link)) for patients with colon cancer.

Macrophages were generated from CD14⁺ monocytes that were isolated from HLA-A2 negative buffycoat PBMCs with CD14 MACS beads (Miltenyi) and differentiated for 6 days in the presence of 50 ng/ml M-CSF (R&D systems). Macrophages were harvested and seeded at 3x10e5 c/well in a 24 well plate for adherence. After 18 hours macrophages were infected with Mtb H37Rv from a log phase culture at a MOI of 10 for 1 hour followed by three washing steps with culture medium in the presence of gentamycin (Lonza Benelux BV, Breda, the Netherlands) (2 times with 30 μg/ml and once with 5 μg/ml). Infected cells were rested overnight, the next day T-cell clones were added at an E:T ratio of 5:1 in duplicate in the presence of specific peptide. After 24 hours of co-culture, the cells were lysed and serial dilutions were plated on 7H10 agar plates, supplemented with BBL Middlebrook OADC enrichment (BD Biosciences) for Mtb CFU determination. Colonies were counted after two to three weeks incubation at 37°C. Specific intracellular Mtb growth inhibition was calculated per experiment after substraction of the average variation in Mtb CFU in the absence of T-cells, all clones were tested in duplicate in 3–4 independent experiments using unrelated Mf donors and results of these experiments were averaged to obtain the percentage specific Mtb growth inhibition.

Buffy coats of 36 mL EDTA full blood from the osteoporotic and non-osteoporotic patients undergoing total hip replacement were isolated by density gradient centrifugation using LSM 1077 [15 ,18 (link)]. Afterwards, CD14 MACS Beads (Miltenyi Biotech, Germany) were used to separate monocytes from lymphocytes and 1 × 10⁶ cells per well were seeded on a 24 well plate. Monocytes were cultivated for differentiation with osteoclast culture medium (α-MEM, 10% FCS standard, 2% L-glutamine, 1% Penicillin/streptomycin, 20 pg/mL RankL, and 5 pg/mL M-CSF). Experiments started at day 10 of cultivation and were performed in triplicate.