Naive CD4 T cells were isolated from one donor as described above using RoboSep separation. The T cells were CFSE labeled as described above. Dendritic cells were isolated from another donor’s PBMCs by using a pan-DC separation kit (StemCell Technologies, catalog number 19251). The DCs and T cells were mixed at a 1:2 ratio in 96-well U-bottom plates and cultured for 7–10 days at 37 °C. The DCs and T cells were either cultured without any treatment or in the presence of an isotype control, CTLA-4-Ig (Abatacept), α-PD-L1 (BioLegend, catalog number 329715), or α-B7-H7 (Amplimmune, Clone 20C5). All of the antibodies were used at 10 µg/ml. After the culture period, CFSE dilution was measured by flow cytometry as a read out for cell proliferation.
Pandc separation kit
Manufactured by STEMCELL
The PanDC separation kit is a laboratory tool designed to isolate plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs) from human peripheral blood mononuclear cells (PBMCs). The kit utilizes a magnetic separation method to enrich the target cell populations.
Automatically generated - may contain errors
3 protocols using pandc separation kit
1
Modulating T cell proliferation with co-inhibitory molecules
2
Splenocyte Activation Assay with Kasugamycin and PolyI:C
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Single cell splenocytes were plated at the density of 5×106 cells/ml isolated and treated with kasugamycin (2mg/ml or 0.02mg/ml) and PolyI:C (High Molecular Weight, Invivogen,CA) (2μg/ml or 0.02μg/ml) for 6 hours. Mixtures of PolyI:C and kasugamycin were incubated together for 30 minutes at 37°C before addition to cells. In Supplementary Fig. 13 , splenocytes were treated for 12 hours, washed 5 times, stained with cell trace violet (Thermo Fisher, CA), and incubated with splenic DCs isolated using PanDC separation kit (Stemcell Technologies, MA). After 6 hour incubation, cell trace violet dim and negative cells were sorted using an FACSAria (BD, NJ) into RLT buffer for RNA extraction. All in-vitro experiments were conducted with a minimum of three replicates and repeated twice.
3
Splenocyte Activation Assay with Kasugamycin and PolyI:C
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