Immunofluorescence, phytohemagglutinin, and immunodiffusion
Blood was taken in heparin tubes (25 unit/mL) in order to assess their immunological status. Accordingly, we determined the total number of leukocytes and lymphocytes in the peripheral blood of all experimental animals. The number of CD19+ and CD3+ lymphocytes and their subpopulation were determined by immunofluorescent cell staining using antibodies conjugated with FITC, explicitly adapted for experiments with rats. CD3+, CD4+, CD8+, and CD19+ FITC monoclonal antibodies were acquired from GALTAG Laboratories (5791 Van Allen Way Carlsbad, CA 92008 USA). Stained cells were examined with a fluorescent microscope (EVOSTM FL, Thermo Fisher Scientific). Surveillance was conducted in a darkened room. We determined both the inhibition reaction of leukocyte migration (LMIR) with phytohemagglutinin (PHA-M, GibcoTM) [23], as well as the concentration of circulating immune complexes (CIC) [24]. We determined the content of immunoglobulins A, M, G, using radial immunodiffusion on Mancini agar gel [25]. We also carried out the determination of the phagocytic activity (PA) of neutrophils [26] and performed nitro blue tetrazolium tests (NTT) [27]. Obtained results were statistically processed; the differences were evaluated using the t criterion of the Student’s t -test.