CD8+ polyclonal or OT-I T cells were isolated from lymph nodes of 6–8-week-old mice using EasySep CD8 negative selection kits (STEMCELL Technologies). B cells were isolated from spleens of 6–8-week-old mice using EasySep B cell negative selection kits (STEMCELL Technologies). In specified experiments, B cells were treated with 1 μg/ml LPS (Sigma-Aldrich) for 5 h before use. BMDCs were generated from treating cultured bone marrow cells with GM-CSF (granulocyte-macrophage colony-stimulating factor) for 7–11 days. IL-4 was added for the last 2 days with 1 μg/ml LPS stimulation 6–24 h before use. BMDCs and B cells were incubated with 1–100 ng/ml SL8 OVA peptide (SIINFEKL) (Anaspec), or variant peptides N4, T4, Q4H7, V4 (gift from E. Palmer and D. Zehn) for at least 30 min at 37°C and washed 3 times.
Easysep b cell negative selection kits
Manufactured by STEMCELL
The EasySep B cell negative selection kits are laboratory products designed to isolate untouched, highly purified B cells from a variety of sample types, such as peripheral blood, cord blood, and bone marrow. The kits use a fast and easy-to-use negative selection approach to enrich for B cells without directly labeling them.
Automatically generated - may contain errors
2 protocols using easysep b cell negative selection kits
1
Isolation and Stimulation of T cells and APCs
2
Isolation and Stimulation of T cells and APCs
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CD8+ polyclonal or OT-I T cells were isolated from lymph nodes of 6–8-week-old mice using EasySep CD8 negative selection kits (STEMCELL Technologies). B cells were isolated from spleens of 6–8-week-old mice using EasySep B cell negative selection kits (STEMCELL Technologies). In specified experiments, B cells were treated with 1 μg/ml LPS (Sigma-Aldrich) for 5 h before use. BMDCs were generated from treating cultured bone marrow cells with GM-CSF (granulocyte-macrophage colony-stimulating factor) for 7–11 days. IL-4 was added for the last 2 days with 1 μg/ml LPS stimulation 6–24 h before use. BMDCs and B cells were incubated with 1–100 ng/ml SL8 OVA peptide (SIINFEKL) (Anaspec), or variant peptides N4, T4, Q4H7, V4 (gift from E. Palmer and D. Zehn) for at least 30 min at 37°C and washed 3 times.
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