Easysep direct human cd4 t cell isolation kit

Blood from BCG treated T1D patients was collected in purple top tubes (K₂EDTA anti-coagulant) and CD4 + T cells were isolated using magnetic EasySep Direct Human CD4 + T Cell isolation kits from Stem Cell Technologies (Vancouver, BC, Canada, Cat# 19662), following the instructions of the manufacturer as previously described^{12 (link),20 (link),25 (link)}. Briefly, 1200 μL of Isolation Cocktail and 1200 μL of Rapid Spheres were mixed with 24 mL of whole blood in a 50 mL centrifuge tube and incubated for 5 min at room temperature. PBS (24 mL) was then added and the tube mixed and placed into an “Easy 50” magnet (Stem Cell Technologies, Cat# 18001). This immobilized the unwanted non-CD4 cells at the side of the tube. After 10 min, the CD4-enriched cell suspension was then transferred into a new tube and the magnetic separation process repeated for 5 min with 1200 uL of fresh Rapid Spheres. The cell suspension was then transferred into a new tube and purified for a third time using the magnet. The resulting final CD4 + T cell preparations had purities of > 90% (Supplementary Fig. S1).

CD4 T cells were isolated from blood from purple tops using magnetic EasySep Direct Human CD4+ T Cell isolation kits from Stemcell Technologies (Vancouver, BC, Canada, Cat# 19662), following the instructions of the manufacturer. Briefly, 1200 μL of Isolation Cocktail and 1200 μL of RapidSpheres were mixed with 24 mL of whole blood in a 50 mL centrifuge tube and incubated for 5 min at room temperature. Twenty-four milliliter of PBS was then added and the tube placed into a “Easy 50” magnet (Stemcell Technologies, Cat# 18001). This immobilized the unwanted non-CD4 cells at the side of the tube. After 10 min, the CD4-enriched cell suspension was then transferred into a new tube and the magnetic separation process repeated for 5 min with fresh Isolation Cocktail and RapidSpheres. The resulting highly CD4 enriched cell suspension was transferred into a new tube and purified for a third time using the magnet. The resulting final CD4 cell preparations had purities of >95%.

Monocytes were isolated directly from whole blood by immunomagnetic negative selection using the EasySep™ Direct Human Monocyte Isolation Kit (Stemcell Technologies, Vancouver, Canada) according to the manufacturer’s instructions.
CD4+ T cells were isolated directly from cord blood of term infants by immunomagnetic negative selection using the EasySep™ Direct Human CD4+ T Cell Isolation Kit (Stemcell Technologies, Vancouver, Canada). Subsequent CD25 depletion was performed using the EasySep™ Human CD25 Positive Selection Kit (Stemcell Technologies, Vancouver, Canada) according to the manufacturer’s instructions.