AF488-labeled nanoparticles were delivered to PBMCs as described earlier. Lymphocytes and monocytes were distinguished based on their forward-scatter and side-scatter characteristics (Figure S13 ). For confocal microscopy samples, 1 × 106 PBMCs per well were seeded in 900 μL of complete RPMI medium in a 24-well plate. For each well, Lipofectamine-complexed and AF488-labeled RNA cubes were added to cells at a final concentration of 10 nM as described earlier. The next day, cells were removed and washed with 1× HBSS. For wheat-germ agglutinin Alexa Fluor 594 (WGA 594) (Molecular Probes, Eugene, OR) staining, cells were fixed for 15 min in 4% formaldehyde and then washed and stained with 1 μg/mL WGA 594 in HBSS for 10 min at room temperature. For the labeling of acidic vesicles, cells were stained with Lyso-ID Red (Enzo Life Sciences, Farmingdale, NY). Lyso-ID Red staining solution was prepared by diluting the assay buffer provided in HBSS and adding 1:1000 Lyso-ID Red stock and 10% FBS. Lyso-ID Red staining was performed for 30 min. After staining with either WGA 594 or Lyso-ID Red, cells were washed twice with HBSS, and slides were prepared using a CytoSpin centrifuge. Images were acquired on a Nikon A1R+ confocal microscope (Nikon Instruments, Melville, NY).
Lyso id red
Manufactured by Enzo Life Sciences
Lyso-ID Red is a fluorescent dye that can be used to detect and visualize lysosomes in live cells. It is a membrane-permeable dye that selectively stains acidic organelles such as lysosomes. The dye exhibits a red fluorescence upon binding to the acidic environment within lysosomes.
Automatically generated - may contain errors
2 protocols using lyso id red
1
Confocal Microscopy of Nanoparticle Uptake
2
Antibody Panel for Vesicular Trafficking
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Antibodies to caveolin-1 (D46G3), clathrin HC (D3C6), Rab5 (C8B1), APPLI (D83H4), EEA1 (C45B10), Rab9A (D52G8), Rab11 (D4F5) and syntaxin-6 (C34B2) rabbit antibodies were from Cell Signaling Technology. MITO-ID Red, Golgi ID Green, ER ID Red and Lyso ID Red were purchased from Enzo Life Sciences. Anti-α3 integrin (CD49c) and APC-anti-α3 integrin (CD49c) antibody (clone ASC-1), anti-CD9 antibody and FITC anti-CD9 antibody (clone H19a), PE anti-human IGF-1R (clone 1H7/CD221), PE anti-human CD115 (clone 9-4D2-1E4), PE anti-human EGFR (AY13), anti-CD59 antibody and PE anti-CD59 antibody (p282), anti-CD276 antibody and APC anti-CD276 antibody (MIH42), anti-CCR2 antibody (K036C2) and anti-CXCR4 antibody (12G5) were from Biolegend. Anti-CXCR2 antibody and APC anti-CXCR2 (Clone 6C6) were from BD Biosciences. Anti-TIMP3 antibody was from Biorbyt Ltd. (Cambridge, United Kingdom). Alexa 555 goat anti-IgG H+L antibodies were from Life Technologies. Alexa 647 anti-P2Y11 (505214) and Alexa 647 anti-MRGX2 (477533) were from R&D Systems. Anti-human LL-37 antibody (1C12) was from Hycult Biotch. Goat anti-mouse IgG (H&L):15 nm Gold was from BBI Solutions. LL-37 was synthesized by the standard FMOC chemistry, purified by reversed phase HPLC, and the mass verified by nanospray mass spectrometry. The concentration was determined by amino acid analysis (Zhang et al., 2008 (link)).
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