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.