Anti cd69 apc

This was undertaken according to our previously published methods (33 (link)). The method utilized whole blood analysis with red cell lysis using Easylyse (BD Biosciences) and with anti-CD56 PE, anti-CD16 FITC, anti-CD3 PE Cy5 and anti-CD69 APC (all supplied by BD Pharmingen). Flow cytometric analysis was undertaken on a Facscalibur (BD Biosciences), and using Cellquest Pro software. A minimum number of 10,000 cells were counted.

Small intestinal mucosal cells were isolated, and the epithelial layer cells were purified separately using 2 mM ethylenediaminetetraacetic acid (EDTA) (1.5 mg/mL collagenase type II (Gibco) in Ca²⁺- and Mg²⁺-free Hank’s balanced salt solution, respectively, as described by Hurasato et al. [34 (link)]. These cells were stained with anti- major histocompatibility complex MHC II PE (phycoerythrin) (BD Pharmingen, clone AF6-120.1), anti-CD11b FITC (fluorescein isothiocyanate) (BD Pharmingen, clone M1/70), anti-CD11c (BD Pharmingen, clone HL3), and anti-CD103 APC (allophycocyanin) (BD Pharmingen, clone M290).
The spleens were removed from all the experimental mice at day 1 after the last immunization. The spleens were perfused with PBS-1x, and then the cell suspension was centrifuged at 300× g and resuspended in RPMI-1640 + 10% FBS for 5 min. The pellets were washed, counted in a hematologic chamber, and stained with anti-CD45 FITC (BD Pharmingen, clone 30-F11), anti-CD4 PE (BD Pharmingen, clone RM4-5), and anti-CD69 APC (BD Pharmingen, clone H1.2F3). Data were acquired using a FACSVerse flow cytometer (BD Biosciences) and analyzed using the software FlowJo 7.6.1.

To compare the surface expression of CD16, CD69, and CD161 on NK cells at the whole range and single-cell level, PBMCs of healthy donors were pre-incubated with cytokines for 12 h and then washed with PBS. All samples were stained with anti-CD3 eFluor 450 (ebioscience, clone UCHT1), anti-CD16 APC-Cy7 (BD, clone 3G8), anti-CD56 PE-Cy7 (BD, clone B159), anti-CD69 APC (BD, clone FN50), and anti-CD161 PE-Cy5 (BD, clone DX12). All antibodies were purchased from BD Biosciences (San Diego, CA, USA) except anti-CD3 from eBiosciences (San Diego, CA, USA). NK cells were defined as phenotype of CD3⁻CD56⁺ and the frequencies of CD16, CD69 and CD161⁺ NK cells were analyzed. CD16/CD69/CD161 expressions on NK cells were acquired on BD FACS Fortessa (BD Biosciences, USA) and then analyzed by FlowJo software (Treestar, Ashland, OR, USA).
The prepared samples were also run on ImageStream χ MarkII system (Amnis Corporation, Seattle, WA, USA) to acquire the data of CD16/CD69/CD161 expressions on single-cell. Twenty thousand events were collected for each sample and single-color control was used to create a compensation matrix to correct for spectral overlap. Collected data were analyzed using IDEAS 3.0 software (Amnis Corporation, Seattle, WA, USA).

To analyze cell surface expression levels of the various markers, the cell suspensions (1 × 10⁶) were incubated with following mAbs: anti-CD3-APC, anti-B220-FITC, anti-CD4-PE, anti-CD4-FITC, anti-CD8-FITC, anti-NK1.1-PerCP.Cy5.5, anti-PLZF-PE, anti-CD44-FITC, anti-CD69-APC, anti-CD62L-PE, anti-CD62L-PErCP, anti-CD25-APC, anti-CD122-PE, and anti-CD127-PE (all BD Pharmingen) acquired in a FACScan (Becton Dickinson, Mountain View, CA, USA) and analyzed using FlowJo software.

Healthy cultures of Jurkat cells with cell density between 0.25 and 1×10⁶ cells/mL were maintained in culture for several weeks. Prior to electroporation, a recovery plate was prepared using pre-warmed RPMI 1640 supplemented with 20% fetal bovine serum and 100 ug/ml of L-glutamate. Jurkat cells were pelleted via centrifugation, then resuspended in 100ul of 1SM buffer (as per [19 (link)]) and 2ug of plasmid and brought to room temperature. Cells were then placed into a 0.2cm electroporation cuvette (Cat#1652086, Bio-Rad, USA) and electroporated using a Lonza Nuceleofactor Electroporator using X4 settings. Cells were then immediately transferred to the recovery plate. Cells were allowed to recover for 4 hours prior to analysis or use in an assay. Post-recovery Jurkat cells and target cells were then resuspended in complete RPMI and placed in 96-well plates at varying ratios of Jurkat and target cells as shown in the text. If adherent target cells were used, cells were first treated with Accutase (Cat#A1110501, Thermo Fisher, USA) to create a single cell suspension. Plates were incubated at 37°C, 5% CO₂ for either 16 to 40 hours. Plates was then stained with anti-CD69 APC (Cat#340560, BD Bioscience, USA) at 0.25ul per well in no-wash format. Plates were then incubated in the dark at RT for 15 minutes and analyzed via flow cytometry using a BD Fortessa device (BD Bioscience, USA).

Anti-CD25-APC-Cy5, anti-CD127-Alexa-Fluor700, anti-CD4-FITC or Pacific blue, anti- CD45RA-FITC or PE-Cy5, anti-CCR7-PE, or PE-Cy7, anti-CD8-PerCP, anti-CD3-AmCyan, anti-IFN-gamma-FITC or APC-Cy7, anti-CD69-APC, anti-PD-1-PE and anti-PD-L1-PECy7, anti-FoxP3-PE were all products of BD Biosciences. Cells were analysed by FACSCanto II, and LSR II (BD Immunocytometry systems). At least 20 000 CD4 or CD8-gated events, and at least 100000 CD4 or CD8-gated events were collected for cell surface and intracellular studies, respectively.