Anti human cd71 fitc

Cells were fixed with 2 % paraformaldehyde and permeable reagent (eBioscience) for 30 min on ice. Cells were stained with fluorescently labeled antibodies (1 μg/ml; 1 h at room temperature), assayed by flow cytometry (FASCAria II, BD Bioscience, Franklin Lakes, NJ, USA), and data were analyzed by Flowjo7.6 software (Tree Star, Inc. Ashland, OR, USA). Primary antibodies used for staining were anti-human cd71-FITC (eBioscience), mAb to HCV core Antigen (Abcam), and goat anti-mouse Alexa555 antibody (Abcam).

All flow cytometric data were acquired on a fluorescence-activated cell sorter (FACS) Fortessa flow cytometer (BD Biosciences) and analyzed using the Flowjo software (Tree Star). All stainings were carried out in FACS buffer (2 mM EDTA and 5% FBS in phosphate-buffered saline (PBS)) for 40 min at room temperature unless otherwise described. Samples were washed twice with FACS buffer prior to flow analyses. The following are the antibodies used at 1:100 dilution: anti-human CD235A-APC (eBioscience, 17-9987042), anti-human CD71-FITC (eBioscience, 11-0719-42), anti-human CD71-PeCy7 (Affymetrix, 25-0719-42), anti-human CD117-PeCy7 (eBioscience, 25-1178-42), anti-human CD117-BV605 (BioLegend, 313217), anti-human CD49e-APC (BioLegend, 328012), anti-human CD29-PerCP-eFluor 710 (Affymetrix, 46-0299-41), anti-human CD49d-PE (Affymetrix, 12-0499-42), anti-human CD240DCE-APC (Miltenyi Biotec, 130-104-818), anti-human CD238-APC (Miltenyi Biotec, 130-104-951), anti-human CD47-PerCP-eFluor 710 (Affymetrix, 46-0479-42), anti-human CD147-PE(Affymetrix, 12-1472-42), anti-human CD59-APC (Affymetrix, 17-0596-42), anti-myc tag-PE (Cell Signaling Technology, 3739), anti-mouse Ter119-APC (eBioscience, 17-5921-83), and anti-mouse CD71-PE (Affymetrix, 12-0711-83). Hoechst 33342 (Life Technologies, H1399) was used to visualize nuclei.

All flow cytometry data were acquired using either using LSR II SORP or LSR Fortessa flow cytometers (BD Biosciences). All staining was carried out in FACS buffer (2% FBS in PBS) for 30 min on ice unless otherwise described. The following antibodies were used anti-human CD235a-APC (eBioscience, Clone HIR2), anti-human CD71-FITC (eBioscience, Clone OKT9), anti-human CD71-PEcy7 (eBioscience, Clone OKT9), ant-human CD49d-PE (Miltenyi, Clone MZ18-24A9), anti-human CD41a-PE (eBioscience, Clone HIP8), anti-human CD11b-PE (eBioscience, Clone ICRF44), anti-mouse Ter119-APC (eBioscience, Clone TER119), anti-mouse CD71-PE (eBioscience, Clone R17217) and Alexa Fluor-647 anti-phospho STAT5 (pY694) (BD Bioscience Cat#: 612599). Hoechst 33342 (Life Technologies, H1399) was used to visualize nuclei.