Alexa fluor 700 mouse anti human cd19

Lipo/siRNA complexes (100pmols of siRNA) marked with rhodamine were incubated with PBMC cultured in 48-well culture plates for 4 and a half hours. Paraformaldehyde-fixed cell suspensions were located on microscope slides as described above. Subsequently, samples were stained with primary purified mouse anti-human CD3 antibody (BD Biosciences 1:30, incubated for 1 hour at RT) followed by secondary antibody goat anti-mouse Cy-5 conjugate (Invitrogen 1:100, incubated for 1 hour at RT). WGA conjugated to Oregon Green® 488 (Invitrogen 1:200,) and 1 μg/ml Hoechst 33342 (Invitrogen) were used to counterstain plasma membrane and nucleic acid respectively. In additional stainings the following antibodies were used: FITC (fluorescein conjugated) mouse anti-human CD56 (BD Bioscience 1:30, incubated for 1 hour at RT); APC (allophycocyanin conjugated) mouse anti-human CD86 (BD Bioscience 1:10, incubated for 1 hour at RT); Alexa Fluor 700 mouse anti-human CD19 (BD Bioscience 1:30, incubated for 1 hour at RT); FITC goat anti-mouse secondary antibody (Invitrogen 1:500, incubated for 1 hour at RT); 1 μg/ml Hoechst 33342 (Invitrogen) was used to counterstain nuclei.

Cells were fixed in 1% paraformaldehyde and when necessary, permeabilized with Perm Buffer III (558050, BD Biosciences, San Jose, CA), and blocked with FcR Blocking Reagent (130-059-901, Miltenyi Biotec, San Diego, CA). Antibodies used in flow cytometry analysis were: biotin mouse anti-human CD49e (555616, BD Biosciences) with streptavidin-APC (SA1005, Caltag, Buckingham, MK18 1TF), alexa fluor 647 mouse anti-fibronectin (563098, BD Biosciences), alexa fluor 700 mouse anti-human CD19 (557921, BD Biosciences), APC-Cy7 mouse anti-human CD11b (560914 BD Biosciences), PE mouse anti-human CD3 (9515-09, Southern Biotechnology, Birmingham, AL), APC mouse anti-human CD36 (561822, BD Biosciences). For lipid uptake analysis, HCS LipidTOX Red Neutral Lipid Stain (Invitrogen), Low Density Acetylated Lipoprotein from Human Plasma conjugated with Alexa Fluor 594 (Invitrogen). Cells were analyzed on the LSR II (BD Biosciences) or Accuri C6 (BD Biosciences) flow cytometers. Data were analyzed with FLOWJO software (Treestar, Ashland, OR, USA).