Cells were resuspended in 1-2 mL of FACS Buffer and 1 L of propidium iodide and sorted utilizing a CytoFLEX SRT cell sorter (Beckman Coulter, Brea, CA) using lymphocyte, singlet, viability, and GFP+ and FITC gates into a tube with Leibovitzs L-15 medium adjusted to catfish tonicity. T cells or natural killer cells Rabbit Polyclonal to ANXA2 (phospho-Ser26) (45), hematopoietic stem and progenitor cells (HSPC)/megakaryocytes (MK) (66), myeloid/epithelial cells (40), and plasma cells (32). The B cell clusters were further defined as different populations of mature B cells, cycling B cells, and plasma cells. The plasma cells highly expressedighmand we demonstrated that the secreted form of the transcript was largely being expressed by these cells. This atlas provides insight into the gene expression of IgM+immune cells in channel catfish. The atlas is publicly available and could be used garner more important information regarding the gene expression of splenic immune cells. Keywords:single-cell RNA sequencing, single-nuclei RNA sequencing, B cell, plasma cell, T cell, lymphocyte, natural killer-like, hematopoietic stem and progenitor cells == 1. Introduction == In modern agriculture, there is a drive to improve the sustainability of production systems due to population Vortioxetine growth and limitations in resources (1,2). Catfish production is the primary aquaculture sector in the United States and in 2023 the industry generated $437 million in sales (3). The two main cultured species are channel catfish (Ictalurus punctatus) and channel x blue catfish (Ictalurus furcatus) hybrids. The primary cause of production losses are pathogenic diseases, particularly the bacterial diseases motileAeromonassepticemia, columnaris disease, and enteric septicemia (4,5). These diseases impact the sustainability and efficiency of the catfish production systems and producers require new disease control methods and interventions. In addition to industry significance, channel catfish are also an important comparative model of the teleost immune system (610). Therefore, there is interest in a further understanding of the catfish immune system. The primary systemic organs of the teleost immune system such as channel catfish are the head kidney, thymus, Vortioxetine and spleen (11). The head kidney (also known as the anterior kidney) has a function equivalent to mammalian bone marrow (11). It maintains stem cells that differentiate into erythrocytes and immune cells (11). The thymus, similarly to the mammalian thymus, trains and matures T cells (11). The spleen is a secondary lymphoid tissue, and the main function of the organ is to provide an environment for lymphocytes, B cells and T cells, and other immune cells; filter the blood of antigens and pathogens; and maintain erythrocyte homeostasis (12). B cells are adaptive immune agents that detect antigens through the B cell receptor (BCR) (13). Antigen binding, along with a second signal, most notably from T helper cells, activates B cells to differentiate into antibody secreting cells, plasmablasts, and plasma cells (13,14). The spleen and B cells are implicated in two of the key pathogenic diseases of catfish. The channel catfish spleen, after exposure in challenge trials, harbor virulentAeromonas hydrophila(vAh) (15), the pathogen that causes motileAeromonassepticemia, andEdwardsiella ictaluri(16), the pathogen that causes enteric septicemia of catfish (ESC). Vortioxetine In addition, yellow catfish (Pelteobagrus fulvidraco) hybrid challenged withA. hydrophilawith vAh had altered B cell gene expression in the head kidney when compared to the control (17). Single-cell/single-nuclei RNAseq (sc/snRNAseq) enables the characterization of gene expression at the individual cell level, which is highly desirable for the research of homogenous tissues. This contrasts with bulk RNAseq, where the transcriptome averages the gene expression across multiple cells and cell types. This means that rare cell types and lowly expressed genes are often masked within the data (18). sc/snRNAseq enables these rare cell types to be discovered and individual cells to be profiled based on their gene expression. Previously, our group developed a single nuclei transcriptomic atlas of theI. punctatusspleen (19). Atlases have also been generated to study zebrafish (Danio rerio) (20) and Atlantic salmon (Salmo salar) (21) spleen. Additionally, scRNAseq was used to understand the effects ofA. hydrophilainfection on the innate immune system of hybrid yellow catfish (P. fulvidraco Pelteobagrus vachelli) sampled from Vortioxetine the head kidney (17). We set out to produce a single nuclei transcriptomic atlas of channel catfish splenic B cells to understand the underlying biology of these.