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Staphylococcus aureus protein binding in human serum

ABSTRACT: Overnight cultures of S. aureus were diluted to OD600 = 1 and 1 ml of culture was resuspended in 500 μl of either PBS, human plasma, or human serum, and incubated rotating at 37 °C for 30 m. Suspensions were washed 3x with PBS supplemented with 500 40% sucrose and 20 mM Sodium Azide. Cells were incubated with immobilized trypsin (ThermoFisher 20230), suspended in PBS supplemented with 500 40% sucrose and 20 mM Sodium Azide, at 37 °C for 2 h. cells were pelleted, and the supernatant containing protein fragments was analyzed by mass spectrometry to determine proteins.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Staphylococcus Aureus

SUBMITTER: Marcus Wittekind

LAB HEAD: Ronan Carroll

PROVIDER: PXD038927 | Pride | 2023-04-14

REPOSITORIES: Pride

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Project description:In this study transcriptome profiling of dendritic cell subtypes was performed using various human dendritic cells. Monocyte-derived DC: CD14+ monocytes were obtained from buffy coats by Ficoll - gradient centrifugation and immunomagnetic cell separation using anti-CD14-conjugated microbeads. Monocytes were resuspended into 6-well culture dishes at a density of 1.5 x 10 million cells/ml and cultured in RPMI 1640 supplemented with 10% FBS containing 800 U/ml GM-CSF and 500 U/ml IL-4. Cells were cultured for 5 days and the IL-4 and GM-CSF addition was repeated at day 2. Dermal DC (DDC) and Langerhans Cells (LC) from skin: Human skin specimens were obtained from healthy donors undergoing corrective breast or abdominal plastic surgery after informed consent. Thick slices (3 mm) of skin containing the epidermis and the dermis were cut by use of a dermatome. Slices of skin were cut in pieces of 1 cm2 and incubated with Dispase II for 30–60 min at 37°C. The epidermis and dermis were separated with tweezers and washed with PBS. To isolate LC, the epidermal sheets were incubated with PBS with 0.05% trypsin for 10 min at 37°C, and a single-cell suspension was prepared by pushing the tissue through 100 µm pore nylon cell strainers with a plunger of a 2-ml syringe. Epidermal cell suspension was enriched for LC by density centrifugation over Lymphoprep and CD1a-guided MACS (Miltenyi Biotec). To isolate DDC, the dermis was incubated with PBS containing Dispase and Collagenase A at 37°C for 2 h, after which, a single-cell suspension was prepared by pushing through 100 µm pore nylon cell strainers with a plunger of a 2-ml syringe. Cell suspension was enriched for DDC by CD1a-guided MACS. Blood Cd1c+ DC: Blood CD1c+ DC were isolated from periferal blood. BDCA1+ (CD1c) DCs were incubated with a lineage-specific PE-labeled Ab mixture, HLA-DR-allophycocyanin, and BDCA1-FITC. Lineage-negative and HLA-DRhigh DCs, positive for BDCA1 were sorted on a FACS and collected in tubes containing 1 ml of 100% FBS. A total of 40,000–100,000 cells was isolated (>98% pure).

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Staphylococcus aureus protein binding in human serum

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