Flow cytometric analysis of peripheral blood lymphocyte subset light scatter characteristics as a means of monitoring the development of rat small bowel allograft rejection

Abstract

SUMMARYThis investigation used flow cytometry to monitor peripheral hlood lymphocyte morphology after rat small bowel transplinitation. Preliminary studies demonsirated that in vitro activated peripheral blood lymphocytes exhibited increased cell size and granularity as measured by flow cytometric analysis of forward (FSc) and side (SSc) light scatter characteristics. The formation of distinct 'activated" light scatter regions by such lymphoblastoid transformation occurred concomitantly with up-regulaied p55IL-2R expression. Heterotopic small bowel transplantation was performed between PVG donor and DA recipient rats without immunosuppression. Animals receiving isografts served as controls. Peripheral blood lymphocyte subsets were identified using appropriate MoAbs. and the light scatter characteristics of each cell subset were determined by backgating strategies. Increased proportions of activated tt/ii T ceil receptor (TCR)-positive cells could be detected inallografted animalsasearly as day 2 post-transplantation. B cells showed peak activation by day 4. at which time the proportion of activated cells was over two-fold greater than that seen in untransplanted animals few activated B cells were detected in isografted animals. Resting natural killer (NK) cell light scatter regions only partially overlap with those of resting T and B lymphocytes, but in allografted animals almost the entire NK population fell outside the resting lymphocyte gate by day 2 post-transplantation, an activation state which was maintained until day 4. These findings associate peripheral blood cell subset lymphoblastoid transformation wilh developing small bowel allograft rejection. Importantly, changes were detected early and prior to the onset of overt rejection. These data suggest that analysis of peripheral blood lymphocyte light scatter properties may provide an insight into in vivo immune status after small bowel transplantation.

Influences of age and sex on leukocytes of healthy horses and their ex vivo cytokine release

“…This indicates that individual animals, although clinically healthy, show up with a different baseline activation or priming of circulating cells. This kind of variability was also reflected by a considerable variation in the size and granularity of circulating leukocytes, which may relate to in vivo encountering of modulating molecules, as shown in other contexts (Webster et al, 1995;Schuberth et al, 2001;Böhmer et al, 2011).…”

“…Flow cytometric methods have been employed to determine the size and granularity/complexity of immune cells to evaluate morphological changes. Even subtle effects on circulating cells can be detected by this method ex vivo and in vitro (Webster et al, 1995;Schuberth et al, 2001;Böhmer et al, 2011).…”

Oral administration of the milk casein-derived tripeptide Val-Pro-Pro attenuates high-fat diet-induced adipose tissue inflammation in mice

“…After washing, fluorescence intensity was measured from cell fractions containing 5000 cells using a FACSCalibur (BD Biosciences), and data were analysed with FlowJo software (Tree Star, Inc.). When fluorescence activity was detected, data were obtained from monocytes using gating definitions, as reported previously ( 14 – 16 ) .…”

Research Article Microdeletion of the AZFc locus with high frequency of mosaicism 46,XY/47XYY in cases of non obstructive azoospermia in eastern population of India

“…5-AzaC (1µg/mL) act as DNA inducer in the lymphocyte was added to the cultures to increase the cell proliferation rate (mitotic index) and to improve the quality of chromosome morphology since the infertile idiopathic males have poor endocrine function and slow cell proliferation index (Saxena et al, 2007). Flowcytometry analysis was performed for the study of cell -kinetics and the 1x10 6 cells (lymphocytes) were collected from cultures (Webster et al, 1995;Darzynkiewicz et al, 2010). The lysis of red blood cells was induced by the addition of RBC lysis buffer for 10 min at 37°C and spun at 900x rpm for 5 min.…”