αcd4 percp cy5

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.

As CD107a is a degranulation marker, αCD107a-FITC (BD), was added together with the Golgi inhibitors (BD) prior to incubation. After stimulation, samples were washed in PBS, stained with LIVE/DEAD Violet Fixable Dead Cell Stain (Invitrogen) for 10 min, washed with PBS, and then stained with αCD3-APC Alexa Fluor 750 (Beckman Coulter), αCD4-PerCP Cy5.5 (BD), αCD14-PacBlue (BD), CD16-PacBlue (BD), αCD19-PacBlue (Invitrogen) and αCD45RO-PE Cy5 (BD) for 25 min, washed twice with FACS buffer (PBS with 2% FBS and 0.01% sodium azide), fixed and permeabilized for 10 min using Cytofix/Cytoperm and Perm/Wash Buffer (BD). The cells were then stained with IFNγ-Alexa Fluor 700 (Invitrogen), IL2-APC (BD), TNFα-PE Cy7 (BD), and perforin-PE (Diaclone) for 30 min and washed twice with Perm/Wash Buffer. Stained samples were stored in 1% formaldehyde at 4°C until flowcytometric analysis (performed within 48 hours). All staining steps and incubations were performed at room temperature.

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).