Small intestines were harvested, rinsed with PBS and Peyer’s Patches were removed. Epithelium associated DCs and T cells were released by incubating for 15 min two times in a 37°C rotating incubator in HBSS media (BioWhittaker, Walkersville, Maryland) containing 5 mM EDTA as previously described48 (link). After a third incubation with HBSS media with 5 mM EDTA, isolation of splenic and lamina propria (LP) cellular populations was performed as previously described48 (link). Cellular suspensions were then passed through a 50µm nylon filter and the cells were prepared for flow cytometric analysis or sorting. Antibodies used for analysis are listed in supplemental table I . DC subpopulations in the SI or APCs in the colon were identified as 7AAD−, CD45+, CD11c+, MHCII+ and either CD103+ or CD103− for flow cytometric sorting. To isolate goblet cells, small intestinal or colonic epithelium was released by incubating for 15 minutes in 37 °C rotating incubator in HBSS media (BioWhittaker, Walkersville, Maryland) containing 5 mM EDTA as previously described. Goblet cells were identified as CD45−CD24−CK-18+UEA-1+, while IECs were identified as CD45−CD24−CK-18−UEA-1−, and sorted directly into RLT buffer (Qiagen, Valencia, CA, USA) for RNA extraction.
Hbss media
Manufactured by Lonza
HBSS media is a balanced salt solution designed for use in cell culture and related laboratory applications. It provides a standardized saline environment to support the maintenance and growth of cells in vitro. The formulation is optimized to maintain appropriate osmolarity, pH, and ionic balance for cell cultures.
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Lab products found in correlation
3 protocols using hbss media
1
Isolation of Intestinal Immune Cells and Goblet Cells
2
Isolation of Intestinal Immune Cells and Goblet Cells
3
Isolation of Intestinal Immune Cells and E. coli
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Small intestines or colons were harvested, rinsed with PBS, and Peyer’s patches or colonic patches were removed and discarded. Epithelial cell populations were released by incubating the intestines for 15 min in a 37 °C rotating incubator in 20 mL Hank’s balanced salt solution (HBSS) media (BioWhittaker) containing 5 mM EDTA and gentamicin (50 μg/mL) as previously described (50 (link)). Following removal of epithelium, isolated lamina propria was cut into pieces. We recovered splenic, MLN, and lamina propria cells as previously described (50 (link)). Lamina propria pieces, spleen, MLN, liver, or stool were homogenized in 500 μL PBS with 200 mg 0.1 mm diameter zirconium silica beads (BioSpec), and homogenized Bullet Blender Tissue Homogenizer (Next Advance) vortexed on bead beater. Supernatant was plated on MacConkey agar containing 20 ng/mL nalidixic acid to identify nalidixic acid-resistant E. coli, and identity of E. coli strain was confirmed through MLST identification.
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