Splenocyte Isolation

The spleen serves as a rich source of lymphocytes. The spleen is the source used most often as it is easily obtainable. Lymph nodes and peripheral blood are also occasionally used as lymphocyte sources. In humans, peripheral blood is often the only source used, unless the spleen or some lymph nodes were being surgically removed (and thus available). 

---

The 48-h concanavalin A-induced lymphoblastogenic responses in splenocytes isolated from BALB/c mice treated with Corynebacterium parvum and 1,1-dimethylhydrazine mice were significantly increased in comparison with C. parvum treatment alone (Frazier et al., 1992), indicating that 1,1-dimethylhydrazine can overcome certain types of immunosuppression. 

Antisense ODNs, small ODNs (15-30 mers) that are complementary to the sequence of pre- or mature mRNA sequences, have been shown to inhibit specifically the expression of numerous genes [128], Abdou et al. reported the use of -CyD bearing galactose moiety for antisense phosphodiester-ODN delivery into cells [129]. In addition, Zhao et al. found inhibitory effects of 6 and 5 on immune stimulation by immunostimulatory phosphorothioate-ODN containing the CpG motif in splenocytes isolated from GDI mice and in vivo [130]. 

Beluga whale, Delphinapterus leucas isolated blood leukocytes and splenocytes exposure to various concentrations of PCBs 138, 153, 169, or 180 for 48 h... 

Immunological Effects. The effects of carbon tetrachloride on the immune system have not been evaluated in humans. Immune responses were not affected in rats orally exposed to carbon tetrachloride (Smialowicz et al. 1991). Parenteral exposure of animals to carbon tetrachloride has been reported to impair the immune system (Kaminski et al. 1989 Muro et al. 1990 Tajima et al. 1985), and oral exposure caused depletion of lymphocytes, hemorrhage, and hemosiderin deposition in the pancreaticoduodenal lymph node (Doi et al. 1991). These findings are supported by in vitro studies in which the IgM antibody formation response of isolated mouse splenocytes to sheep erythrocytes was inhibited in a dose-dependent manner when the splenocytes were exposed to carbon tetrachloride for 1-3 hours in the presence of cocultured hepatocytes (Kaminski and Stevens 1992). No effects were observed in the absence of cocultured hepatocytes. Mice appear to be more sensitive than rats to carbon tetrachloride-induced immunosuppression, but the biological significance to humans of these reported effects are yet ascertainable from the available data. 

Fig. 5.1 Technique to produce monoclonal antibodies. After mice are injected with the antigen, the splenocytes are isolated and fused with myeloma cells. The fused cells are cultured (1) and the supernatants are assayed for the presence of desired antibodies (2). The cells from antibodypositive wells are cloned (3) and supernatants are tested for the desired antibodies (4) followed by expansion of the positive clones (5) and in vitro or in vivo propagation to produce the needed amount of monoclonal antibodies (see Color Insert)...

Isolated cells from infected mice are less proliferative upon in-vitro activation and increase inducible nitric oxide synthase expression in peritoneal cells, yet splenocytes have normal cytokine responses to ConA (Dai and Gottstein, 1999)... 

Starting with the second week of nevirapine treatment, progressive infiltration of T cells into the rat skin dermis was observed. To assess the specific role of the T cell populations in triggering the onset of skin rash, Shenton et al. transferred splenocyte T cells from rechallenged rats into naive recipients. Spleen CD4 T and CD8 T cells were isolated from the nevirapine-rechallenged rats, purified, and intravenously injected into naive recipients, which were then started on a full (150 mg/kg/day) nevirapine dose (Shenton et al. 2005). Recipients of CD4 T cells developed skin rash 9 days later, while CDS" T cell recipients behaved as nevirapine naive rats, only developing red ears by day 7 and skin rash by day 21... 

Matrix metalloproteinase-9 (MMP-9) is a secreted multidomain enzyme, which plays an important role in the migration of immime cells. In a recent study, it is reported that fucoidan posttranslationally regulated MMP-9 secretion from U937 (Jintang et al., 2010). Fucoidan isolated from U. pinnatifida possesses immunomodulating activity to produce cytokines and chemokines from macrophages and splenocytes (Yoo et al., 2007). 

Because we are dealing with a relatively large number of cells (1-3 x 10 splenocytes), we recommend first isolating total RNA from the entire spleen, followed by mRNA isolation (see Protocol 3). 

We recommend using the TBU REAGENT to isolate undegraded total RNA from splenocytes. This procedure is an improvement of the guanidine thiostep extraction procedure described by Qiomczynski and Sacchi (5). 

Immtmoassay systems using monoclonal antibody (MAh) against natural products have become an important tool for studies on receptor binding analysis, enzyme assay, and quantitative and/or qualitative analytical techniques in animals or plants [75]. Immunogenic forskolin-bovine serum albumin (BSA) conjugate was synthesized via 7-deacetyl-7-hemisuccinyl forskolin. BALB/c male mice were injected intraperitoneally with forskolin-BSA conjugate emulsified with Fretmd s complete adjuvant. Splenocytes were isolated from the hyperimmunized BALB/c mice and fused with the P3-X63-Ag8-Ul myeloma cells. Six hybridomas producing monoclonal antibodies (MAbs) reactive to forskolin were obtained. 

The polysaccharide isolated from B. kummingense showed an immunomodulatory action on the proliferative response of mouse splenocytes to mitogens as measured by H-thymidine incoporation [24]. 

The level of IL-4 in the spleen did not change after the same treatment. Instead, in the liver the oral administration of polysaccharides stimulated the secretion of both IFN-y and IL-4 at a dose of 200 mg/kg, together with a significant proliferation of hepatocytes [63]. Polysaccharide extracted from mucilage of D. huoshanense exhibited an effect in murine splenocytes. It induced the production of several cytokines, including IFN-y, IL-10, IL-6, and IL-la, and hematopoietic growth factors GM-CSF and G-CSF in mice splenocytes [64]. Further experiments were performed in mice and human cells in-vitro on polysaccharides isolated from D. huoshanense to point up the potentiality of these biopolymers in a therapeutic approach to some immime disease [65],... 

Four polysaccharides (FCp-1, FCp-2, FCp-3 and FCp-4) were isolated from citrus fruits (Citrus medica L. var. sarcodactylis) after hot-water extraction and ethanol precipitation. Among all, only the FCp-3, a polysaccharide with a molecular weight of 177.1 KDa, showed an immunological activity evaluated by splenocyte and thymocyte proliferation assay. FCp-3 displayed a significant splenocyte proliferation at a dose of > 25 microg/mL, and showed a moderate effect on thymocyte proliferation at the same dose [70]. 

DNA origami tube CpG ODN - Isolated mouse splenocytes—free uptake Schuller et al. (2011)... 

---