Anti pe macs beads

Lymphocytes were isolated from spleens of C57BL/6 mice. We used anti-mFoxp3-PE to stain the cells and anti-PE MACS beads (Miltenyi Biotec) to isolate them. Contaminating CD8⁺ T cells and B cells were removed via negative depletion using Dynal beads. Pure (up to 95%) Foxp3⁺CD4⁺ T cells (Tregs) were labeled with CFSE (5 μM), a new dye for fluorescent labeling of living cells, and injected i.p. into TLR2^–/–mice (2 × 10⁶ per mouse). After four hours, the mice were challenged with F. nucleatum (10⁸ CFU per day) for a period of 1 week. The splenocytes were stained with anti-Foxp3-PE and anti-CD4-APCs to confirm that the CFSE⁺ cells were still Foxp3⁺. The production of proinflammatory cytokines in the blood of the mice was measured.

T cell suspensions were prepared from lymph nodes of OT-1 or polyclonal C57/BL6 mice. Where stated, PE-conjugated Abs were used for purification of naive CD8 T cells by negative selection. Lymph node cells were incubated with anti–CD4-PE (eBioscience) and anti–I-A/I-E-PE (BioLegend) followed by a 15-min incubation with anti-PE MACS beads (Miltenyi Biotec). Cells were added to MACS LS columns (Miltenyi Biotec) and nonbinding CD8 T cells were collected. Wild-type (WT) and Cav1-KO lymph node CD8 T cells were cultured in IMDM media (Invitrogen) supplemented with 5% FCS, l-glutamine, antibiotics, and 50 μM 2-ME. OT-1 CD8 T cells were activated by the addition of peptides SIINFEKL (N4), SIITFEKL (T4), or SIIGFEKL (G4) in the presence of CD3ε-KO spleen cells as APCs to culture media as indicated. At the end of culture, CD8 T cells were stained with the relevant Abs, run through a MACSQuant flow cytometer (Miltenyi Biotec), and analyzed with FlowJo software (Tree Star). For proliferation assays, OT-1 T cells were labeled with CellTrace Violet (CTV; Molecular Probes) for 10 min before washing and culture. Proliferation was determined by assessing CTV dilution using FlowJo software (Tree Star). For LFA-1 blocking assays, M17/4 mAb (eBioscience) was incubated in 40-μl suspensions of 100,000 cells in 96-well round-bottom plates and incubated at 37°C/5% CO₂.