Flow cytometric characterization of peripheral blood leukocyte populations of 3 neotropical snake species:Boa constrictor, Bothrops jararaca, andCrotalus durissus

Abstract

Based on FSC and SSC, 4 distinct leukocyte populations were found in ficoll or percoll density gradient isolated leukocytes from peripheral blood from boa and viper species. Further optimization of the technique should allow the performance of functional assays.

Telomere length varies substantially between blood cell types in a reptile

“…For each individual sample, the telomere-associated FITC fluorescence of lizard blood cells was reported relative to that of an internal standard (co-processed and co-analysed control CCRF-CEM cells). Cell types were identified based on size using forward scatter area (FSC-A) and cellular morphology (specifically granularity; figure 1 ) using side scatter area (SSC-A) with excitation at 488 nm and comparison to previously published identifications ([ 27 , 35 ]; figure 2 ).

Figure 1.

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“…This kit does not suffer from the interaction of sub-telomeric sequences, unlike methods such as telomere restriction fragment analysis using southern blotting (TRF), and the probe hybridizes with telomere repeat sequences (TTAGGG) typical of vertebrates including lizards. The resulting fluorescence intensity of the cells is directly correlated with the length of the telomeres [26][27][28]. This method therefore provides a relative indication of telomere length between different cells.…”

“…Images were captured and analysed using a Leica SP8 confocal microscope (Wetzler, Germany) and Leica Application Suite X software (version 3.4.2). Cell types were identified based on size, cellular morphology and comparison to published Giemsa stained cells [27,28,[34][35][36]. Following this literature, we conservatively identified three cell types, red blood cells (RBCs), lymphocytes (T or B cells, not differentiated) and the reptile-specific azurophils (a reptilian monocyte; [21]).…”

“…Even if (most) of these cell types do not go through mitosis once generated from haematopoietic stem cells, telomere attrition rates could vary profoundly, depending on exposure time to reactive oxygen species (ROS) and other reactive molecules that affect attrition [5,25]. Therefore, to assess to what extent different cell types in the same individual and blood sample have different telomere length, we set out to identify blood cells of different types using flow cytometry and simultaneously measure the lengths of their telomeres using the same technique [26][27][28].…”