Cd44 apc clone im7

Cells were submitted to a mild trypsinisation with a solution of 0.125% trypsin and 0.78 mM EDTA (Sigma-Aldrich, St. Louis, MO, USA). Cell detachment from the culture flask was monitored under an inverted microscope, and the enzymatic reaction was immediately inhibited by adding culture medium supplemented with 10% FBS. The cells were centrifuged at 1200 rpm for 5 min at 4 °C, the pellet was resuspended in culture medium supplemented with 10% FBS, and cells were quantified in a hemocytometer with trypan blue to evaluate viable cells. For FACS, approximately 5 × 10⁵ cells were washed twice with cold PBS containing 3% FBS and 0.1% sodium azide (FACS buffer) and incubated for 30 min on ice with the following directly conjugated antibodies: mouse anti-human CD29-Alexa 700 (clone TS2/16), CD51 R-PE (R-phycoerythrin, clone NKI-M9), CD49b-FITC (fluorescein, clone P1E6-C5), CD51/CD61-FITC (clone 23C6) from Biolegend and CD24-PE (clone SN3, Thermo Fischer, Waltham, MA, USA) and CD44-APC (clone IM7, eBioscience, San Diego, CA, USA). The cells were washed twice with FACS buffer. Acquisition was performed on a FACSCanto-II (BD Biosciences, Franklin Lakes, NJ, USA) using the software Diva 6.1.3 (BD Biosciences, Franklin Lakes, NJ, USA). Data were analysed using Diva and FlowJo (Tree Star Inc., Ashland OR, USA).

Spleen tissue was gently mashed through a 70 µm nylon membrane filter in lymphocyte separation medium to obtain single cell suspensions. Lymphocytes were collected following centrifugation. Following cell count and dilution, lymphocytes were treated with anti-mouse CD16/CD32 antibodies (Mouse BD Fc Block, BD Biosciences) for 15 min and then stained with CD4-FITC (clone RM4-5, eBioscience), CD8-PerCP-Cy5.5 (clone 53-6.7, eBioscience), CD62L-PE (clone MEL-14, BD Biosciences), and CD44-APC (clone IM7, eBioscience) antibodies. The intracellular staining of TLR8 (clone 112H7.15, Dendritics) or BCL2 (clone BCL/10C4, Biolegend) was performed according to the instructions of the manufacturer (Fixation/Permeabilization Solution Kit, BD Biosciences). Following staining, cells were washed with PBS, fixed in 1% formaldehyde, and analyzed on BD Accuri C6 Plus or FACSAria (BD bioscience). Frequency of target cell types was calculated by FlowJo software.