Lymph nodes Sample Clauses

Lymph nodes. Stromal cells were isolated from mediastinal lymph nodes of mice receiving heart grafts using a technique described by Xxxxxx et al. (218) with some modifications. Lymph nodes were harvested into a 40mm petri dish containing 1ml LEC digestion media, and the capsules disrupted using a 27G needle attached to a 1ml syringe. The tissue was then transferred to a 5ml polypropylene round-bottomed tube, and washed with a further 1ml LEC digestion media to ensure all tissue was transferred. The tubes containing lymph node tissue were kept on ice for transportation between the biological services unit and laboratory. Xxxxxx was allowed to settle at the bottom of the tube and the supernatant was aspirated using a sterile pastette to a sterile 50ml Falcon tube with 100µm nylon mesh attached. The mesh was washed with LEC digestion media, and the tube placed on ice. To the lymph node tissue, 750µl digestion mix 1 (LEC digestion media plus 1mg/ml collagenase IV (Worthington Biochemical Corporation, Lakewood, NJ, US) and 40µg/ml DNAse 1 (Roche Diagnositcs, Basel, Switzerland)) pre-warmed to 37ºC was added, and the tube incubated at 37ºC for 30 minutes with 30 seconds gentle vortexing every 10 minutes. Xxxxxx was again allowed to settle at the bottom of the tube and supernatant aspirated to the 50ml Falcon tube with 100µm nylon mesh attached. The mesh was washed with LEC digestion media, and the tube placed on ice. To the lymph node tissue, 750µl digestion mix 2 (LEC digestion media plus 3.5mg/ml collagenase D (Roche Diagnositcs) and 40µg/ml DNAse 1) pre-warmed to 37ºC was added, and the tube incubated at 37ºC for 10 minutes. The tissue was further disaggregated by pipetting up and down using a 1000µl pipette set at 700µl for approximately 2 minutes or until no visible tissue fragments remained. The digested tissue was then transferred to the 50ml Falcon with 100µm mesh attached. The mesh was washed 5 times with 1ml LEC digestion media, and the tube placed on ice. The tube containing digested lymph node tissue was centrifuged at 1500RPM for 5 minutes at 4ºC. The supernatant was discarded and the lymph node cells were re- suspended in FACS buffer.
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Lymph nodes k. Neurological

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