Aria cell sorter cytometer

Human Peripheral Blood Mononuclear Cells (PBMCs) were obtained by Ficoll density gradient centrifugation (Eurobio^AbCys) and split for tetramer staining into tubes containing 200x10⁶ cells. Cells were incubated with 200 μL PE-, APC and BV421-conjugated tetramers diluted to 10 μg/mL in PBS plus 2% FBS for 30 min at room temperature and then washed with 15 ml ice-cold sorting buffer (PBS plus 0.5% BSA plus 0.2% EDTA). The tetramer-stained cells were then enriched using anti-PE and APC Ab-coated immunomagnetic beads [20 (link)]. The resulting enriched fractions were stained with anti-human CD3-BV510 (BD Biosciences) and anti-CD19-PerCpCy5.5 (BD Biosciences) mAbs. Stained samples were then collected on an ARIA Cell Sorter Cytometer (BD Biosciences) and single CD19⁺ CD3⁻ PE⁺ APC⁺ BV421⁻ tetramer cells were collected in individual PCR tubes containing 10 μL of 1X PBS, 10 mM DTT (Thermo Fischer Scientific) and 10 units of RNAse Out (Thermo Fischer Scientific) per well. Single cells were frozen at -80 °C immediately. The total number of tetramer-positive B cells was divided by the total number of B cells within the starting PMBC sample to calculate the frequency of pMHC-specific B cells. Counting beads (Dako) were used to normalize results.

B cell isolation was performed as previously described [8 , 10 (link)]. Briefly, PBMCs were obtained by Ficoll density gradient centrifugation and incubated with PE-, APC and BV421-conjugated tetramers (10 μg/mL in PBS plus 2% FBS, for 30 min at room temperature). The tetramer-stained cells were enriched using anti-PE and-APC Ab-coated paramagnetic beads and then stained with anti-CD19-PerCpCy5.5 (BD Biosciences) mAbs. Stained samples were collected on an ARIA Cell Sorter Cytometer (BD Biosciences) and single CD19⁺ CD3⁻ PE⁺ APC⁺ BV421⁻ tetramer cells were collected in individual PCR tubes.