Single-cell suspensions of spleen and mesenteric LNs (mLNs) were prepared as previously described (Yeh et al., 2011 (link)) and resuspended in R10 medium (RPMI 1640 with 10% FCS, 2 mM l -glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 1× nonessential amino acids, 1 µM sodium pyruvate, and 50 µM β-mercaptoethanol). Large intestine lamina propria lymphocytes were isolated using the Lamina Propria Dissociation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Cells were then isolated by centrifugation over a Percoll gradient, washed, and resuspended in R10 media.
Protocol detail
Isolation of Lymphocytes from Spleen, mLN, and Intestine
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Amino acids
Cells
Centrifugation
Glutamine
Lamina propria
Large intestine
Lymphocytes
Mercaptoethanol
Mesenteric
Mlns
Penicillin
Percoll
Pyruvate
Sodium
Spleen
Streptomycin
Corresponding Organization : University of Alabama at Birmingham
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Variable analysis
independent variables
- Tissue type (spleen, mesenteric lymph nodes, large intestine lamina propria)
- Not explicitly mentioned
- R10 media composition (RPMI 1640 with 10% FCS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 1× nonessential amino acids, 1 µM sodium pyruvate, and 50 µM β-mercaptoethanol)
- Cell isolation method (centrifugation over Percoll gradient)
- No positive or negative controls were explicitly mentioned in the input protocol.
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