Nhs rhodamine

Four-to-six weeks-old female BALB/c mice were used for the isolation of peritoneal macrophages59 (link). Macrophages were plated onto a culture chamber at 2 × 10⁵ cells/well. Hc yeasts were labelled with 40 μg/mL of NHS Rhodamine (Thermo Scientific, Rockford, lL, USA) for 30 min at 25 °C and washed (3X) with excess of PBS. Cells were incubated with the distinct Cn-gly or PBS as described above. Following incubation, cells were washed, suspended in DMEM, enumerated, and added to the macrophages in a 5:1 (yeast:macrophage) ratio. Plates were incubated for 1 h in 5% CO₂ atmosphere. After three washes with PBS, yeasts were stained using 0.5 mg/mL of Uvitex 2B to distinguish internalized versus extracellular yeasts. Wells were washed (3X) with PBS and fixed with a 4% formaldehyde solution in PBS. The number of macrophages and yeasts were recorded for each field by microscopic enumeration and at least 200 macrophages were counted. The percentage of phagocytosis was determined as the ratio of macrophages with internalized yeast cells divided by total macrophages, and the phagocytic index as the average number of yeast inside macrophages55 (link).

Spatiotemporal analysis using HS-AFM was performed using the BIXAM real-time imaging system (Olympus Corp., Tokyo, Japan), as described previously [29 (link),31 (link),32 (link),33 (link)]. Prior to HS-AFM, P. gingivalis cells were labeled with the fluorescence dye rhodamine (excitation/emission of 543/580 nm) using NHS-rhodamine (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. For immobilization, rhodamine-labeled cells were incubated on glass slides (SF17370, Matsunami glass, Osaka, Japan) for 5 min. The commercially available cantilever BL-AC10DS-A2 (Olympus Corp.) and USC-F0.8-k0.1 (Nanoworld AG, Neuchâtel, Switzerland) was used for analysis.

The primary antibody against SNX9 (OTI1E4, 1:1000) was purchased from Origene, anti-Pacsin (PA5-83983, 1:200), anti-N-WASP (PA5-52198, 1:200) and anti-β-actin (MA5-15739, 1:2000) antibodies were sourced from Thermo Scientific and the anti-actin (#7301-01, 1:500) antibody was from Hypermol. Anti-penta-His (#34660, 1:2500) and anti-GST (#2622, 1:1000) antibodies were obtained from Qiagen and Cell Signaling, respectively. Antibodies against Cpn0677 were generated by Eurogentec (Belgium, 1:50 in immunofluorescence).
Secondary anti-rabbit, anti-rat and anti-mouse antibodies coupled to Alexa488 or Alexa594 (2 µg/ml) or coupled to alkaline phosphatase (1:10,000) were purchased from Thermo Scientific. Rhodamine-Phalloidin (#R415) was purchased from Thermo Scientific. All lipids used in this study were obtained from Avanti Lipids, NHS-FITC and NHS-Rhodamine and MitoTracker™-Red from Thermo Scientific. Wiskostatin (W2270-5MG) was purchased from Merck. G-actin (#8101-01), G-actin-Atto647 (#8158-03) and Arp2/3 (#8413-01) were obtained from Hypermol.

In total, 50 μm coronal sections were sliced and collected using a vibratome (Leica, VT1200S) and first treated with 1% hydrogen peroxide for 30 min, then incubated in 0.5% blocking buffer (Roche, Mannheim, Germany—Cat 11096176001) in 1x Tris/NaCL buffer for 1 h at room temperature. Sections were incubated overnight at 4 °C with a rabbit anti-c-fos primary antibody (1:5000 dilution, Synaptic Systems, Goettingen, Germany) and then with a secondary antibody (peroxidase conjugated donkey-anti-rabbit 1:500, Jackson Immunoresearch, Baltimore, PA, USA) for 1 h. For the final tyramide signal amplification and labeling step, sections from the hM4Di group were incubated for 30 min in NHS-fluorescein (1:500, Thermo Fisher Scientific, MA) sections from the eGFP group for 30 min in NHS-rhodamine (1:500, Thermo Fisher Scientific, MA) diluted in 0.1 M borate buffer with 0.01% hydrogen peroxide. Sections were washed in PBS before and after all procedures (5 × 5 min), which took place on a shaker and at room temperature. Following staining, brain slices were mounted on gelatin-coated slides and air dried before being coverslipped with Fluoroshield Mounting medium with DAPI for nuclear staining (Abcam, MA).

Tetraethyl orthosilicate (TEOS), cetyltrimethylammonium bromide (CTAB), 3-(trihydroxysilyl) propyl methylphosphonate, and aminopropyltriethoxy silane (APTS) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Branched-polyethylenimine (PEI, 10 kDa) was purchased from Alfa Aesar (Ward Hill, MA, USA). Maleimide-PEG (5 kDa)-NHS was purchased from JenKem Technology USA (Plano, TX, USA). Trastuzumab (Herceptin®, Genentech) and cetuximab (Erbitux®, Eli Lilly) were obtained from the OHSU pharmacy (Portland, OR, USA). Phosphate-buffered saline (PBS) (pH 7.2) was obtained from Life Technologies (Carlsbad, CA, USA). Zeba spin desalting columns (MW 40 kDa), RNase-free water, Traut’s reagent, ethanol, HCl, NHS-rhodamine, and sodium hydroxide were purchased from Thermo Fisher Scientific (Waltham, MA, USA). All reagents were of the highest purity grade available. Cell lines (MDAMB468, BT549, MDAMB231, KPL4 and MCF7) were obtained from American Type Culture Collection and maintained following the supplier’s recommendation.