Inflamed mucosa (n = 3 AFRS nasal polyps, n = 1 mucosa adjacent to mycetoma) and control mucosa from inferior turbinates (n = 4) were each taken from the same patient and prepared as previously decribed.5 (link) Briefly, tissue was incubated for 5 hours at 37°C in Dulbecco’s Modified-Eagle Medium (Lonza, Basel, Switzerland) with 5μg/mL Brefeldin A (Sigma-Aldrich, St. Louis, MO). Tissue was enzymatically digested releasing single cells into suspension.5 (link) Ethylenediamine tetra-acetic acid (EDTA) (Sigma-Aldrich, St. Louis, MO) at final concentration 2mM was added to quench the digestion. Debris was removed using a 40-μm cell strainer and red blood cells (RBCs) were removed using an RBC lysis buffer (Miltenyi Biotech, Bergisch Gladbach, Germany). Cells were serially washed in 2% BSA in PBS (Sigma-Aldrich, St. Louis, MO) for analysis by flow cytometry.
Protocol detail
Isolation of Immune Cells from Inflamed Nasal Mucosa
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Dulbecco’s Modified-Eagle Medium
Brefeldin A
Ethylenediamine tetra-acetic acid (EDTA)
RBC lysis buffer
PBS
Acetic acid
Brefeldin a
Buffer
Cells
Digestion
Eagle
Ethylenediamine
Flow cytometry
Mucosa
Mycetoma
Nasal polyps
Patient
Tetra
Tissue
Turbinates
Corresponding Organization : Monell Chemical Senses Center
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Variable analysis
independent variables
- Inflamed mucosa (n = 3 AFRS nasal polyps, n = 1 mucosa adjacent to mycetoma)
- Control mucosa from inferior turbinates (n = 4)
- Not explicitly mentioned
- Tissue was incubated for 5 hours at 37°C in Dulbecco's Modified-Eagle Medium (Lonza, Basel, Switzerland) with 5μg/mL Brefeldin A (Sigma-Aldrich, St. Louis, MO)
- Ethylenediamine tetra-acetic acid (EDTA) (Sigma-Aldrich, St. Louis, MO) at final concentration 2mM was added to quench the digestion
- Debris was removed using a 40-μm cell strainer
- Red blood cells (RBCs) were removed using an RBC lysis buffer (Miltenyi Biotech, Bergisch Gladbach, Germany)
- Cells were serially washed in 2% BSA in PBS (Sigma-Aldrich, St. Louis, MO) for analysis by flow cytometry
- The inflamed mucosa (AFRS nasal polyps and mucosa adjacent to mycetoma) and control mucosa from inferior turbinates were taken from the same patient, providing a within-subject control.
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