Cd44 pecy7 clone im7

The cells were harvested and stained with properly titrated fluorochrome-conjugated monoclonal antibodies (as described before [28] (link)). In brief, cells were stained with antibodies against luminal cytokeratin's (CK) conjugated to PE (mixture of cytokeratin 8, 18 -clone C11- and cytokeratin 19 -clone A53-B/A2-, Veridex LCC, Raritan, NJ), CD24-FITC (clone SN3, eBiosciences, San Diego, CA), CD44-PeCy7 (clone IM7, eBiosciences) and EpCAM-FITC (clone EBA-1, BD Biosciences, San Jose). CK staining was preceded by a fixation and permeabilization step using FIX & PERM reagents (An Der Grub Bio Research GmbH, Austria). Protein expression was measured on a FACSCanto flow cytometer (BD Biosciences). The signal-to-noise ratio (s/n) was calculated (i.e. geometric mean fluorescence intensity (MFI) of the stained population divided by the MFI of the unstained population) to correct for auto fluorescence.

Splenocyte populations of vaccinated mice were obtained at 7 days post-boost and were assessed by flow cytometry, as previously described, using the following directly conjugated Abs, CD3 PerCP-Cy5.5 (clone17A2; Biolegend, San Diego, CA, USA), CD4 APC/Cy7 (clone GK1.5; eBioscience, San Diego, CA, USA), CD8 APC (clone 53–6.7; eBioscience, San Diego, CA, USA), CD44 PE-Cy7 (clone IM7; eBioscience, San Diego, CA, USA), and CD62L Alexa Fluor 488 (Clone MEL-14; Biolegend, San Diego, CA, USA). Subsequently, cells were washed and fixed on ice with 2% paraformaldehyde in PBS and the number of CD4 and CD8 T effector cells was then quantified using an LSRII flow cytometer.