Lamina propria cells from colonic tissue were harvested as before8 (link). For intracellular cytokine staining, cells were stimulated using the eBioscience cell stimulation cocktail for 4 h at 37 °C. Cells were fixed and permeabilized using the Biolegend fixation and permeabilization buffer. The following antibodies (clones) were used for staining: CD45 (30-F11), CD19 (6D5), CD11b (M1/70), CD90.2 (53–2.1), CD3 (145–2C11), TCR-β (H57–597), CD4 (RM4–5), CD8 (53–6.7), Gr-1 (RB6–8C5), MHCII (M5/114.15.2), CD11c (N418), CD103 (2E7), IFN-γ (XMG1.2) from Biolegend and IL-22 (1H8PWSR), and IL-13 (eBio13A) from eBioscience. All samples were blocked with Fc block (TruStainfcx) and stained with a fixable live dead stain (Invitrogen). FlowJo v.10 was used to analyze flow cytometry data. Intestinal epithelial cells (IECs) were harvested by incubation at 37°C with 2mM DTT (Sigma) followed by two incubations with 5mM EDTA and then digested with Dispase (Sigma) and DNase (Sigma). IECs were sorted as PI−CD45−EpCam+ using the following antibody clones: CD45 (30-F11) and EpCam (G8.8) from Biolegend on an FACSAria II (BD Biosciences). Purity of IECs was ≥ 95%.
Protocol detail
Isolation and Characterization of Colonic Immune Cells
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cell stimulation cocktail
fixation and permeabilization buffer
IL-22 (1H8PWSR),
IL-13 (eBio13A)
Fc block
fixable live dead stain
DTT
Dispase
DNase
CD45 (30-F11)
EpCam (G8.8)
FACSAria II
Antibodies
Antibody
Buffer
Cd103
Cd11b
Cd90
Cells
Clones
Colonic
Cytokine
Dispase
Dnase
Edta
Epcam
Epithelial cells
Flow cytometry
Ifn γ
Il 13
Il 22
Intestinal
Intracellular
Lamina propria
Stain
Tissue
Corresponding Organization :
Other organizations : New York University, Oregon Health & Science University
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Variable analysis
independent variables
- Cell stimulation using eBioscience cell stimulation cocktail for 4 h at 37 °C
- Intracellular cytokine production (IFN-γ, IL-22, IL-13)
- Cell surface marker expression (CD45, CD19, CD11b, CD90.2, CD3, TCR-β, CD4, CD8, Gr-1, MHCII, CD11c, CD103)
- Intestinal epithelial cell (IEC) isolation and sorting (PI-, CD45-, EpCam+)
- Fixation and permeabilization using Biolegend fixation and permeabilization buffer
- Fc block (TruStainfcx) and live/dead stain (Invitrogen)
- Incubation with 2mM DTT, 5mM EDTA, Dispase, and DNase for IEC isolation
- Not explicitly mentioned
- Not explicitly mentioned
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