Falcon 70 μm cell strainer

Mice were inspected personally for tumor development. Infiltrated lymph nodes, spleen and bone marrow were collected and smashed in PBS. Cell suspensions were passed three times through a Falcon 70 μm Cell Strainer (#352350; Corning), centrifuged (80g for 5 min) and resuspended in 10 ml of Erythrocyte Lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA). After another centrifugation step, cells were resuspended in 10 ml of MACS buffer (PBS, 2 mM EDTA, and 0.5% BSA), and part of the cells used for in vitro culture. Primary cells were grown in suspension in B-cell medium composed of a 1:1 ratio of DMEM (ECM0103L; Euroclone) and IMDM (I3390; Sigma-Aldrich), supplemented with 10% fetal calf serum (Globefarm Ltd.), 2 mM L-glutamine (Invitrogen Life Technologies), 1% non-essential amino acids (NEAAs), 1% penicillin/streptomycin and 25 μM β-mercaptoethanol. A lymphoma cell line was considered as stabilized when the splitting ratio reached 1:10 every 2 d, which usually occurred upon 2 wk of in vitro culture.

Eighteen-day-old embryonated NOVOgen Brown eggs were obtained from a commercial breeder (Verbeek Broederij, Zeewolde, the Netherlands). Chicken embryos were removed from the eggs and euthanized by decapitation. Next, the tibiae and femurs were collected, bone heads were removed, and bone marrow was harvested by flushing the bones with RPMI-1640 cell culture medium supplemented with GlutaMAX™-I, phenol red, and HEPES (Gibco™, Life Technologies Limited, Paisley, UK) under sterile conditions using a Plastipak™ 10-ml syringe with a Microlance™ 3 21-G needle (both from BD Biosciences, Pharmingen, San Diego, CA, USA). Bones and bone marrow cells were kept on ice during the whole procedure. Bone marrow cells from 200 embryos were pooled, gently squeezed through a Falcon® 70-μm cell strainer (Corning®, Corning B.V. Life Sciences, Amsterdam, the Netherlands), and stored at −140°C in RPMI, 50% chicken serum (Gibco™, Life Technologies Limited, Paisley, UK), and 10% DMSO (Honeywell, Bucharest, Romania). This procedure resulted in batches comprising 1.3–2.3 × 10⁹ bone marrow cells, which were frozen at a concentration of 2.5–5 × 10⁷ cells per cryotube.

To isolate cells from the native hearts, cardiac allografts, spleens, and draining lymph nodes, organs were excised, minced, and digested with 500 U/mL collagenase (Roche) for 30 minutes at 37°C, followed by incubation with 0.1 M EDTA in PBS, pH 7.2, buffer for 5 minutes before final suspension in 5 mM EDTA, 1% FBS in PBS, pH 7.2. Isolated cells were then mechanically dissociated through a Falcon 70 μm cell strainer (Corning), and RBCs were lysed using hypotonic ACK buffer (Lonza).