Samples from 35 H pylori-infected donors (10 duodenal ulcer, 1 gastric ulcer, 24 without ulceration) and 46 uninfected donors were analysed by flow cytometry. These groups were 64% and 52% male, respectively; mean ages 51.0 and 53.6 years.
Six gastric biopsy samples were collected into culture medium (RPMI 1640/10% fetal calf serum/100 U/mL penicillin G/100 μg/mL streptomycin sulfate), rubbed through a sterile disposable 100 μm cell strainer (BD Biosciences, Oxford, UK), washed and resuspended at 1×106/mL.9 (link) Peripheral blood mononuclear cells (PBMCs) were purified by density gradient centrifugation using Histopaque-1077 and resuspended at 1×106/mL.
Extracellular staining using anti-CD4-phycoerythrin (PE)-Texas Red (ECD; Beckman Coulter, High Wycombe, UK) and anti-CD25-PE-cyanin 7 (Pc7) with anti-CD127-PE, anti-CD62L-PE (eBioscience, San Diego, USA), anti-integrin αE-PE (eBioscience), anti-CCR10-PE, anti-CXCR1-PE or anti-intergrin β7-PE (eBioscience) and anti-CCR6-Alexa Fluor 647 (A647), anti-CCR7-A647, anti-CCR9-A647 or anti-CXCR2-A647 was carried out before cells were fixed in 0.5% formaldehyde. FOXP3 Perm buffer set (BioLegend) permeabilised cells were stained with anti-FOXP3-Alexa Fluor 488 (A488). Data on 200 000 events were acquired using a Beckman Coulter Cytomics FC500 and analysed with Weasel V.3.0, using appropriate isotype controls.
Six gastric biopsy samples were collected into culture medium (RPMI 1640/10% fetal calf serum/100 U/mL penicillin G/100 μg/mL streptomycin sulfate), rubbed through a sterile disposable 100 μm cell strainer (BD Biosciences, Oxford, UK), washed and resuspended at 1×106/mL.9 (link) Peripheral blood mononuclear cells (PBMCs) were purified by density gradient centrifugation using Histopaque-1077 and resuspended at 1×106/mL.
Extracellular staining using anti-CD4-phycoerythrin (PE)-Texas Red (ECD; Beckman Coulter, High Wycombe, UK) and anti-CD25-PE-cyanin 7 (Pc7) with anti-CD127-PE, anti-CD62L-PE (eBioscience, San Diego, USA), anti-integrin αE-PE (eBioscience), anti-CCR10-PE, anti-CXCR1-PE or anti-intergrin β7-PE (eBioscience) and anti-CCR6-Alexa Fluor 647 (A647), anti-CCR7-A647, anti-CCR9-A647 or anti-CXCR2-A647 was carried out before cells were fixed in 0.5% formaldehyde. FOXP3 Perm buffer set (BioLegend) permeabilised cells were stained with anti-FOXP3-Alexa Fluor 488 (A488). Data on 200 000 events were acquired using a Beckman Coulter Cytomics FC500 and analysed with Weasel V.3.0, using appropriate isotype controls.