αly6g af700

Clinical laboratory improvement amendments certified clinical laboratories were used for testing purposes, which included initial testing for pregnancy or infectious disease (Fig. 1B). Follow-up testing included serum chemistries and flow cytometric analysis of peripheral blood lymphocytes. Cell suspensions were stained and analyzed on an LSRII flow cytometer (BD Biosciences). Cells were gated and identified as follows: CD8 T-cells (CD8⁺), NK cells (CD8⁻, CD4⁻, CD49b⁺), Regulatory T-cells (CD25⁺, FoxP3⁺), monocytic MDSCs (CD45⁺, CD11b⁺, Ly6C^Hi), granulocytic MDSCs (CD45⁺, CD11b⁺, Ly6G^Hi). Antibody conjugates were purchased from BioLegend (San Diego, CA): αCD8/FITC (100705), αCD4/PerCP-Cy5.5 (100539), αCD49b/PE/Cy7 (108921), αCD45.2/BV510 (109837), αCD11b/BV650 (101239) and BD Pharmingen: αLy6C/PerCP/Cy5.5 (560525) and αLy6G/AF700 (561236) and eBioscience (Thermo Fisher Scientific, Waltham, MA): αFoxP3/Alexa Fluor 700 (56-5773-82). Patients receiving doses of 10⁵ through 10⁹ CFU had peripheral blood flow cytometry performed before administration and at 5 weeks post administration. Patients receiving 10¹⁰ CFU had additional peripheral blood flow cytometry performed at 2 and 3 weeks post administration, if possible.

A/J mice were sacrificed 5 days after last treatment. Tumors and spleens were homogenized using a mouse Tumor Dissociation Kit (Miltenyi Biotec) according to manufacturer's instructions. Cell suspensions were stained according to manufacturer instructions. Intracellular staining for FoxP3 was performed using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience: 00-5523).
The stained cells were analyzed on an LSRII flow cytometer (BD Biosciences). Cells were gated and identified as follows: CD8 T-cells (CD8⁺), NK cells (CD8⁻, CD4⁻, CD49b⁺), T-regulatory cells (CD4⁺, CD25⁺, FoxP3⁺), monocytic MDSCs (CD45⁺, CD11b⁺, Ly6C^Hi), granulocytic MDSCs (CD45⁺, CD11b⁺, Ly6G^Hi), and dendritic cell (CD11b⁺, CD11c⁺, CD8⁺). Antibody conjugates were purchased from BioLegend: αCD8/FITC (100705), αCD4/PerCP-Cy5.5 (100539), αCD49b/PE/Cy7 (108921), αCD45.2/PE (109808), αCD11b/BV650 (101239), αPDL1/PE-Cy7 (124313), αPDL1/PE-Cy7 isotype (400617), αCD25/BV650 (102038), BD Pharmingen: αLy6C/PerCP/Cy5.5 (560525) and αLy6G/AF700 (561236), and eBioscience: FoxP3/AF700 (56-5773-80). Dead cells were excluded from analysis using Fixable Viability Dye eFluor 780 (eBioscience 65-0865-14).