Removing the beans from the hot water can be accomplished by draining the beans through a strainer, or by lifting out the blanching basket if you used one.
Single-cell spleen suspensions for flow cytometry were obtained by mincing the tissue through a 70 μm strainer followed by erythrocyte lysis.
Splenocytes and lymph node cells were prepared by dispersion of the organ and passage through a 70-µm cell strainer followed by red cell lysis and resuspension in the media of choice for the given experiment.
Homogenates from both legs of the same mouse were pooled and passed though a 100 µm cell strainer (BD Falcon) by centrifugation at 1100×g for 5 minutes at 4°C to filter residual myofibrillar material from the nuclei.
For isolation of lymphocytes in the lung, lung tissues were ground with fine metal strainer, followed by digestion with HBSS containing 5% FBS, 125 U/mL collagenase I (GIBCO Inc., Grand Island, NY, USA), 60 U/mL DNase I and 60 U/mL hyaluronidase (Sigma, St Louis, MO, USA) for 60 min at 37°C.
A syringe plunger is used to disrupt the pieces through the strainer, followed by washing with the flow-through medium until the strainer is clean.
For purification of splenic DCs and SIGN-R1+ macrophages single-cell spleen suspensions were prepared by treatment with collagenase IV and DNase I for 30 min at 37C and mincing the tissue through a 70 μm strainer followed by erythrocyte lysis.
Cell suspension was filtered through a 40 μM cell strainer, followed by 1500 rpm centrifugation for 5 min and the resulting pellet was resuspended in 1 ml of warm PBS. 1 million of hypothalamic cells from WT and PGC-1β mice were used for each assay.
SMARTA transgenic CD4+ T cells were obtained by processing whole spleens through 70-μm nylon cell strainers followed by separation with anti-CD4 MicroBeads.
After overnight incubation, zoospores and cysts were collected by pouring the culture filtrate through a 40 μm cell strainer and concentrated by centrifugation (5 min: 1500 g).
