Fig 1: Peptide H-HPLN particles bind CD177-expressing human neutrophils in whole blood.Peptide H-HPLN particles were incubated with whole human blood for 1 h at 37°C. The white blood cells were collected from the buffy coat and the red blood cells were lysed. The white blood cells were centrifuged onto glass slides, fixed in methanol and stained with mouse anti-human CD177 antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. Scrambled Peptide H-HPLN particles were used as a negative control. The experiment was carried out four times using blood from four different donors. In each case, slides with several hundred cells were examined and the micrographs are representative of these results. Scale bar, 10 μm.
Fig 2: HPLN particles displaying Peptide M become internalized by CHO cells expressing mouse CD177-HA.CHO cells expressing mouse CD177 containing an HA-tag were incubated with 75 μg/ml Peptide M-HPLN particles for 2 h on ice to allow binding. Cells were then washed and kept on ice or warmed to 37°C for 1 h. Cells were then treated with subtilisin to remove surface-bound Peptide M-HPLN particles, or treated with buffer alone. After fixation and permeabilization, mouse CD177-HA was stained with a mouse anti-HA antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. The experiment was carried out once. Most cells out of more than 100 cells visualized on the slides had a similar staining pattern as those seen in these micrographs. Scale bar, 50 μm.
Fig 3: Time course of CD177 and Peptide H-HPLN redistribution in human neutrophils.Purified human neutrophils were incubated with Peptide H-HPLNs at 37°C with aliquots taken at 0, 15, 30, 60 and 120 min. Cells were rinsed with PBS, centrifuged onto glass slides, fixed in methanol and stained with mouse anti-human CD177 antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. The experiment was carried out once using blood from a donor with a high CD177 expression level. Hundreds of cells were examined and the micrographs are representative of these results. Scale bar, 10 μm.
Fig 4: Peptide M-HPLN particles bind CD177-expressing mouse neutrophils in whole blood.Peptide M-HPLN particles were incubated with whole mouse blood for 1 h at 37°C. The white blood cells were collected from the buffy coat and the red blood cells were lysed. The white blood cells were centrifuged onto glass slides, fixed in methanol and stained with rabbit anti-mouse CD177 polyclonal antibody and Alexa Fluor 488 goat anti-rabbit secondary antibody. Peptide H-HPLN particles were used as a negative control. The experiment was carried out twice. The micrographs represent the results from >100 cells. Scale bar, 10 μm.
Fig 5: Some Peptide H-HPLN particles co-localize with LAMP2 after prolonged incubation, suggesting delivery into the lysosomes.CHO cells expressing human CD177 were incubated for 3 h and 17 h at 37°C with 75 μg/ml Peptide H-HPLN particles. Cells were fixed and permeabilized, and the lysosomes were stained with a mouse anti-hamster LAMP2 antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. The experiment was carried out once. The selected micrographs are representative of more than 100 cells with similar labeling patterns. Scale bar, 50 μm.
Supplier Page from DNASU for CD177 (Homo sapiens) in pDNR-Dual (Creator donor/master vector)