Apc conjugated anti cd4

Human serum, M-CSF, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), acutase, and ovalbumin was from Sigma. Heparin (5000 U/ml) was obtained from Leo Pharma, Denmark and complete protease inhibitor tablets was from Roche.
Proteins: Recombinant wild-type SOD3 and SOD3 containing a C-terminal c-Myc tag (SOD3-cMyc) was produced as previously described [42 (link)]. For the generation of the SOD3-cMyc expression plasmid, we used the forward primer (5′-TATACAGCTAGCATGCTGGCGCTACTGTGTTCC-3′) and a reverse primer encompassing the cMyc tag (underlined) (5′-TATGAATTCTCACAGATCCTCTTCTGAGATGAGTTTTTGTTCGGCGGCCTTGCACTCGCTCTC-3′) and cloned the product into the pIRES vector. The sequence of the obtained plasmid was verified by sequencing. mAb 5G8D4 and 7F6D9 anti-human SOD3 (GenScript) and rabbit anti SOD3 antisera (Davids Biotechnologie) were developed using recombinant human SOD3 as antigen and antibodies recovered by using protein G-Sepharose. Recombinant human and murine IFNγ was obtained from Invitrogen. ovalbumin was from Sigma. Human receptor-associated protein (RAP) was obtained from ENZO life sciences (BML-SE552). HRP-conjugated goat anti-rabbit Ig was from DAKO. Flow cytometric analyses were performed using PE-conjugated anti-CD14 (B&D Biosciences), APC-conjugated anti-CD4 (B&D Biosciences) or APC-conjugated mAb5G8D4 anti-SOD3.

The following monoclonal antibodies (mAbs) were obtained from BD Biosciences (San Jose, CA, USA): fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-Cy7- or allophycocyanin (APC)-conjugated anti-CD3ε (clone 145-2C11); FITC-, PE-Cy7, or APC-conjugated anti-CD4 (clone RM4–5); PE-conjugated anti-CD103 (clone M290); PE-Cy7-conjugated anti-GITR (clone DTA-1); PE-Cy7-conjugated anti-CD11b (clone M1/70); APC-conjugated anti-CD25 (clone PC61); PE-conjugated anti-IL10 (clone JES5-16E3); PE-Cy7-conjugated anti-CD117 (c-kit) (clone 2B8); FITC- or PE-conjugated anti-Foxp3 (clone NRRF-30); PE-conjugated anti-IL17A (clone eBio17B7). The following mAbs from Thermo Fisher Scientific were used: FITC- or APC-conjugated anti-CD19 (clone ID3); FITC- or APC-conjugated anti-Ly-6G (Gr-1) (clone RB6-8C5). FITC- or PE-conjugated anti-IL4 (clone BVD6-24G2); FITC- or APC-conjugated anti-FcεRI (clone MAR-1); PE-conjugated anti-IFNγ (clone XMG1.2). Cells were harvested and washed twice with cold 0.5% BSA-containing PBS (FACS buffer) for staining surface markers. For blocking Fc receptors, the cells were incubated with anti-CD16/CD32 mAbs (clone 2.4G2) on ice for 10 min and subsequently stained with fluorescently labeled mAbs. Flow cytometric data were acquired using a FACSCalibur flow cytometer (Becton Dickson, San Jose, CA, USA) and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).

Peripheral white blood cells were isolated through hemolysis by adding FACS lysing solution (BD Biosciences, San Jose, CA, USA). After the precipitates were washed twice by phosphate-buffered saline (PBS), cells were labelled according to our routine method [14 (link)]. Antibodies for PerCP-Cy5.5-conjugated anti-CD3; APC-conjugated anti-CD4; FITC-conjugated anti-CD45RA; PE-conjugated anti-CD25 and anti-Vδ2; PE-Cy7-conjugated anti-CD28 and anti-NKG2D; BV421-conjugated anti-56, anti-127, anti-CD194, and anti-TCRγδ; BV510-conjugated anti-CD8 and anti-NKP46; Alexa Fluor 647-conjugated anti-CCR7 and anti-CXCR5; Alexa Fluor 484-conjugated anti-CD183, and BB515-conjugated anti-PD-1 were purchased from BD Biosciences.
Samples were run on a BD LSR Fortessa™ cell analyzer (BD Biosciences) at Shuangzhi Purui Medical Laboratory Co., Ltd. (China), and the data were analyzed using FlowJo 10.1 software (Tree Star Inc., Ashland, USA).