EasySep™ Human B Cell Enrichment Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
For positive selection, an aliquot of 10×106 frozen PBMCs was used for sequential isolations of B cells followed by CD8+ T cells and another 10x106 frozen PBMC aliquot was used for sequential isolation of monocytes followed by CD4+ T cells.	according to manufacturer's instructions.

Upstream tips
For positive selection, an aliquot of 10×106 frozen PBMCs was used for sequential isolations of B cells followed by CD8+ T cells and another 10x106 frozen PBMC aliquot was used for sequential isolation of monocytes followed by CD4+ T cells.
Protocol tips
according to manufacturer's instructions.

Publication protocol

For positive selection, an aliquot of 10×106 frozen PBMCs was used for sequential isolations of B cells followed by CD8+ T cells and another 10x106 frozen PBMC aliquot was used for sequential isolation of monocytes followed by CD4+ T cells. All isolations were performed using the following kits from StemCell Technologies, Inc. (Vancouver, Canada): human CD19 positive selection kit (B cells), human CD8 positive selection kit (CD8+ T cells), human CD33 positive selection kit (monocytes), human CD4 positive selection kit (CD4+ T cells) according to manufacturer's instructions. For negative selection, independent aliquots of 10×106 frozen PBMCs were used to isolate CD4+ and CD8+ T cells, B cells and monocytes. EasySep enrichment kits (human CD4+ T cell enrichment kit, human CD8+ T cell enrichment kit, human B cell enrichment kit, human monocyte enrichment kit) from StemCell Technologies, Inc. (Vancouver, Canada) were used according to manufacturer's instructions. Purity was assessed for all cells isolated by positive and negative selection. The following antibodies from BD Biosciences, Inc. (San Diego, CA) were used: CD4-FITC (Multiclone Leu3a+3b), CD14-PE (MoP9), CD8-PE-Cy7 (SK1), CD19-APC (HIB19), CD3-PerCP-Cy5.5 (SK7). For further isolation and purity assessment details, please refer to the Supplemental Methods. Cell suspensions were immediately centrifuged and pellets were resuspended in RLT buffer from RNeasy Mini kit (Qiagen, Inc., Valencia, CA) supplemented with β-mercaptoethanol and stored at −80°C until RNA extraction.

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