CD19+ B Cell Isolation and Sorting

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.

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Devilder M.C., Moyon M., Gautreau-Rolland L., Navet B., Perroteau J., Delbos F., Gesnel M.C., Breathnach R, & Saulquin X. (2019). Ex vivo evolution of human antibodies by CRISPR-X: from a naive B cell repertoire to affinity matured antibodies. BMC Biotechnology, 19, 14.

Publication 2019

anti-CD19-PerCpCy5.5 ARIA Cell Sorter Cytometer

Aria B cell Cell isolation Cells Density gradient centrifugation Ficoll Isolation Mabs Tetramer

Corresponding Organization :

Other organizations : Inserm, Université d'Angers, Centre National de la Recherche Scientifique, Nantes Université, Centre Pays de la Loire

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Variable analysis

independent variables

Incubation of PBMCs with PE-, APC and BV421-conjugated tetramers (10 μg/mL in PBS plus 2% FBS, for 30 min at room temperature)

dependent variables

Enrichment of tetramer-stained cells using anti-PE and-APC Ab-coated paramagnetic beads
Staining of cells with anti-CD19-PerCpCy5.5 (BD Biosciences) mAbs
Collection of single CD19+ CD3- PE+ APC+ BV421- tetramer cells on an ARIA Cell Sorter Cytometer (BD Biosciences)

control variables

Ficoll density gradient centrifugation to obtain PBMCs
Incubation time (30 min) and temperature (room temperature) for tetramer staining
Use of PBS plus 2% FBS as the buffer for tetramer staining

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