We initially identified the population of IgM+ RBCs serendipitously when studying the B lymphocyte population in trout lymphoid organs (Supplementary Figure 3). Splenic and anterior kidney tissues were initially passed through 100-μm cell strainers (Corning, Durham, NC, USA) using a combination of dissociating the tissue with the textured end of a syringe plunger and washing with Iscove’s Modified Dulbecco’s Medium (IMDM, Life Technologies Limited, Paisley, UK). This process began with placing the tissue onto the mesh of the cell strainer. We added 1 mL of IMDM onto the tissue to prevent it from drying. We repeatedly applied gentle force and a twisting motion with the syringe plunger. We then washed the dissociated tissue. Each wash consisted of pipetting 1 mL of IMDM onto the strainer, focusing the wash on areas of the strainer with tissue. Three washes were performed in total. This is how we excluded cellular aggregates larger than 100 μm. For this purpose, the IMDM was not supplemented or added with any fetal bovine serum or antibiotics. We then loaded the cell suspension onto 25% Percoll (one part Percoll diluted in three parts IMDM, no other component was added) (Percoll purchased from Cytiva, Uppsala, Sweden) for centrifugation at 500 g for 15 min (gentle acceleration and braking) to exclude cells of lower density such as adipocytes and fibroblasts. After we washed away the remaining Percoll with excess IMDM (no additives), we counted 10 million cells as input material for the MACS. They were stained with 100 ng of mAb 1.14 specific to salmonid IgM (21 (link), 22 (link)), obtained from Dr. Bernd Köllner (Institute of Immunology of the Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health). The IgM+ cells were positively selected using MACS and anti-mouse IgG microbeads and LS columns (both purchased from Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions, with purity assessed by flow cytometry on a BD FACSCanto II (BD Biosciences, Prague, Czech Republic). All flow cytometry data here and throughout the manuscript were analyzed with the FlowJo v10 software (Becton, Dickinson and Co., Ashland, OR, USA).
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