Nk1.1 pe

Human cervical tissue was obtained from two sets of five healthy women (age range 42–47 years old) undergoing hysterectomy for benign indication at HUGTP. After confirmation of healthy tissue status by the Pathological Anatomy Service, a piece from ecto and endocervix separated by anatomical localization was delivered to the laboratory in refrigerated RPMI 1640 medium (Cellgro, Manassas, VA) containing 10% FBS (Lonza, Basel, Switzerland), 500U/mL penicillin, 500μg/mL streptomycin, 5μg/mL fungizone and 1μg/mL gentamycin (Life Technologies). Tissue was processed within the next 12 h after surgery, and 8-mm³ block-dissection was performed as described [26 (link)]. Tissue digestion of 5–9 pieces of ecto or endocervix with collagenase IV (Invitrogen) was immediately executed as described [26 (link)]. Tissue blocks were then dissociated manually with a disposable pellet pestle and filtered through a 70μm-cell strainer (BD Biosciences). After centrifugation, pellet was suspended in staining buffer (1% mouse serum, 1% goat serum in PBS) and stained with different combinations of CD3-eFluor 605 (eBiosciences), CD14-V450, CD11c-PE-Cy7, CD8-V500, HLA-DR-PerCP-Cy5.5, CD69-Horizon PE-CF594 (BD Biosciences), NK1.1-PE, γδTCR-FITC, Vα7.2-APC-H7 (Miltenyi Biotec), CD103-FITC and Vα24-APC (BioLegend). Data were acquired and analyzed as described for blood.

T cell absolute count was performed on blood samples kept from the mandibular vein of the mouse.
For the phenotypic characterization of cell populations, the following antibodies were used: CD8-FITC (Miltenyi Biotec), CD25-APC (Pharmingen), CD3-PE-Vio770 (Miltenyi Biotec), CD4-APC-Vio770 (Milteny Biotec), CD45R (B220)-Violblu (Milteny Biotec), NK1.1-PE (Milteny Biotec).
At predetermined optimal concentrations, 100 μl of blood was stained by incubation with the antibodies. Fifty microliters of CountBright Absolute Counting Beads (Molecular Probes) was added, and, following lysis of red blood cells, cells were acquired on a CyAn Cytometer (Beckman Coulter). By comparing the ratio of bead events to cell events, absolute numbers of cells in the sample were calculated.
Some experiments were performed by acquiring the stained blood samples on the CytoFLEX cytometer (Coulter), equipped with a volumetric sample injection module, which enables volumetric sampling and provides absolute cell counts for all samples without the use of beads.