scientificprotocols authored over 6 years ago
Authors: Jennifer Gommerman & Olga Rojas
The largest mucosal surface in the body is in the gastrointestinal (GI) tract, a location that is heavily colonized by normally harmless microbes. A key mechanism required for maintaining a homeostatic balance between this microbial burden and the lymphocytes that densely populate the GI tract is the production and trans-epithelial transport of poly-reactive IgA1. Within the mucosal tissues, B cells respond to cytokines, sometimes in the absence of T cell help, undergo class switch recombination (CSR) of their Immunoglobulin (Ig) receptor to IgA, and differentiate to become plasma cells (PC)2. However, IgA-secreting PC likely have additional attributes that are needed for coping with the tremendous bacterial load in the GI tract. Here we describe a detailed method to characterize IgA+B220lowCD11clowiNOS+TNFα+ cells that we named TNFα-iNOS-producing (TiP)-PC in the lamina propria of mice by FACS.
The largest mucosal surface in the body is in the gastrointestinal (GI) tract, a location that is heavily colonized by normally harmless microbes. A key mechanism required for maintaining a homeostatic balance between this microbial burden and the lymphocytes that densely populate the GI tract is the production and trans-epithelial transport of poly-reactive IgA1. Within the mucosal tissues, B cells respond to cytokines, sometimes in the absence of T cell help, undergo class switch recombination (CSR) of their Immunoglobulin (Ig) receptor to IgA, and differentiate to become plasma cells (PC)2. However, IgA-secreting PC likely have additional attributes that are needed for coping with the tremendous bacterial load in the GI tract. Here we describe a detailed method to characterize IgA+B220lowCD11clowiNOS+TNFα+ cells that we named TNFα-iNOS-producing (TiP)-PC in the lamina propria of mice by FACS.
Isolation of lamina propria (LP) cells from small intestine:
Once LP cells are isolated, we proceed to do staining in four main steps: 1. viability staining, 2. surface marker staining, 3.fixation/permeabilization and 4. intracellular staining, as follows:
CAUTION: You should use PBS only (without proteins) as a buffer during viability staining.
12.Incubate at 4 °C for 30 min, protected from the light
13.Wash 2x in FACS Buffer, spin at 1200 rpm for 5 min at 4 °C
14.Resuspend cells in 100 ml/well Cytofix/Cytoperm
15.Incubate for 20 min at 4 °C
16.Wash 2x in 1x Perm/wash (diluted in water)
17.Resuspend in 50 ml of intracellular staining cocktail (prepare the cocktail in Perm/wash buffer):
CAUTION: Spin the cocktail 5000 rpm for 10 min before you use it.
18.Incubate for 20 min at 4 °C
19.Wash 2x in 1x Perm/wash (diluted in water)
20.Re-suspend cells in 200 ml of FACS buffer.
21.Store at 4˚C until ready to run FACS.
22.Acquire samples in a LSRII machine. We use BD Compensation beads for compensation as well as application settings to enhance reproducibilitybetween experiments.
Once isolated, the staining procedure of lamina propria cells takes approximately three hours.
The viability should be over 60%, otherwise the frequency of IgA+iNOS+ may be dramatically decreased since IgA+ PC appear to be very susceptible to processing-related death.
The mean frequency of IgA+B220low cells is around 14%. From this IgA+B220low cells about 3-5% are iNOS+ and 1% are iNOS+TNF+ double-positive, so you need to acquire at least 2 million of total lymphocytes in order to have enough cells to analyze.
We thank Dionne White in the Faculty of Medicine Flow Cytometry core facility. C.P. is supported by a CIHR operating grant MOP# 9862. R.C. is supported in part by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health. A.M. is supported by a CIHR operating grant MOP# 89783. J.H.F. acknowledges support by an APART-fellowship of the Austrian Academy of Sciences, McGill start-up funds and a CIHR operating grant MOP#114972. N.S. acknowledges the support of a CIHR Doctoral Award. J.L.G. is funded by the Canadian Institutes of Health Research (CIHR) and acknowledges the support of CIHR operating grant MOP# 67157 as well as infrastructure support from the Ontario Research Fund and that Canadian Foundation for Innovation.
Complete Protocol with Figures 1-2: Detection of TNF/iNOS in small intestinal lamina propria cells
Download Complete Protocol with Figures 1-2
Acquisition of a multifunctional IgA+ plasma cell phenotype in the gut. Jörg H. Fritz, Olga Lucia Rojas, Nathalie Simard, Douglas D. McCarthy, Siegfried Hapfelmeier, Stephen Rubino, Susan J. Robertson, Mani Larijani, Jean Gosselin, Ivaylo I. Ivanov, Alberto Martin, Rafael Casellas, Dana J. Philpott, Stephen E. Girardin, Kathy D. McCoy, Andrew J. Macpherson, Christopher J. Paige, and Jennifer L. Gommerman. Nature doi:10.1038/nature10698
Jennifer Gommerman & Olga Rojas, Gommerman Lab, University of Toronto
Correspondence to: Olga Rojas ([email protected])
Source: Protocol Exchange (2011) doi:10.1038/protex.2011.267. Originally published online 16 December 2011.