GSM4185143: MG_Human_PMNC_LTA_pep2c_hPMNC_2c_10_3; Homo sapiens; RNA-Seq
Samples were suspended and incubated in staining medium (PBS without calcium and magnesium with 2% heat-inactivated Fetal Calf/Bovine Serum (FCS/FBS) and 1mM EDTA) containing fluorescent antibodies. Following incubation, cells were washed with staining buffer only or staining buffer with DAPI, centrifuged, filtered through 80μm filter and read. Sorting was performed on FACSAria III or FACSAria Fusion (BD Biosciences). Blood was retrieved from healthy participants and added to 60% Dextran solution (6% Dextran T-500 (Sigma) in PBS++, pH 7.4) and 40% Citrate buffer (5% Na Citrate + 1.6% Citric acid in PBS++). To isolate peripheral blood mononuclear cells, mixture was left for 30 minutes in room temperature and supernatant was collected and loaded on Ficoll. Tubes were centrifuged in 920 g in 8oC for 25 minutes without breaks and interphase were collected and washed with cold PBS. Pellet was suspended and filtered with a 70μm filter. Cells were washed with MACS buffer (PBS with 0.5% BSA and 2mM EDTA), counted and incubated in MACS buffer with 1:10 CD14 microbeads (Miltenyi Biotech), 100μl for 107 cells. CD14+ cells were isolated by MACS magnetic column (Miltenyi Biotech) according to manufacturer's instructions. Cells were seeded on 24-well tissue culture plates, 1x105 cells per well in RPMI medium (Biological industries) with 20% heat-inactivated fetal bovine serum (FBS) (Life technologies), 1% penicillin streptomycin amphotericin b solution (Biological industries), 1% L-Glutamine (Biological industries). After 3 days, half the medium was renewed. Cells were analyzed on day 7. RNA-seq of populations was performed as described previously (Lavin 2014)