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Table 1: Age dependent cellular viability (in percentage) (n = 3). Whereas in case of young adults as the volume
as obtained in culture of brain and number of cells obtained from each adult
Groups % of viable cells rat was sufficiently higher 3 individuals (n = 3) were
Early embryo (ED 10 ± 1) 87 ± 3 used for three sets of experiments. In each age group,
Late embryo (ED 18 ± 2) 86 ± 2 cell numbers were diluted to 10 cells/mL. The isolated
7
Neonates (D 5 ± 1) 90 ± 3
Young adult (D 45 ± 5) 92 ± 3 cells along with 0.1% NBT and ultrafine carbon particle
were allowed to incubate at 37 °C overnight. The
voltage. The specimens were scanned and photographed. reaction was stopped with 0.1 N chilled HCl. A sample
of cells from each reaction mixture was transferred to a
Phagocytosis and measurement of oxidative burst of
microglia by nitroblue tetrazolium chloride reduction Neubauer’s chamber to observe under a phase contrast
assay microscope (Nikon Eclipse TS 100) and documented
using a CCD Camera (DS-Fi2-U3) and analysed with NIS
Phagocytic properties of the isolated cells from different Elements BR software (Nikon Corporation, Japan). The
age groups were observed and quantified parallel remainder of each reaction mixture was centrifuged,
by incubating cells with ultrafine carbon particles supernatant removed, pellets resuspended in pyridine,
(SRL, India) and nitroblue tetrazolium chloride and boiled for 10 min, which converted the extract
(NBT) salt (Sigma, USA). NBT reduction assays with into blue by reducing NBT into formazan, which can
carbon particle ingestion, were performed to visualize be measured colorimetrically at 540 nm. The OD value
phagocytosis and measure the oxidative burst of is directly proportional to the level of phagocytosis
phagocytic cells. For both early and late embryos, a or, more precisely, the reactive oxygen species (ROS)
[21]
pool of cells was made from a single mother’s foetus or generation, by different developmental age groups.
pups. In case of early embryos from a single mother, an Statistical analysis
average of 7 embryos were obtained from which cells
were isolated. The same process was done for another Microsoft Excel (Redmond, WA) was used to compute
2 pregnant mothers (n = 3 pregnant mother foetus). For and tabulate experimental results. For statistical
late embryos, an average of 6 embryos were isolated interpretation, one-way ANOVAs, followed by Tukey-
from a single mother for an experiment, which was Kramer post-hoc tests, were performed, and the
conducted in triplicate (n = 3 pregnant mother foetus). results were analysed using OriginPro 8 Software and
In the case of neonates, 3 sets, each containing an graphically represented. Data were expressed as mean
average of 6 pups, were taken to execute the experiment ± SD and a P value < 0.05 was considered significant.

Figure 4: Isolated microglial cells. (A) Microglia isolated from rats shows varied morphology i.e. both irregular and elongated cells with extended pseudopod. (×10); (B)
immunostaining of isolated cells from adult rat brain showing CD11b+ (positivity) prominently with morphological variations, i.e., both irregular and elongated cells are
observed (×10); (C) whereas in case of neonates most of CD11b+ cells are showing irregular morphology (×10)

42 Neuroimmunol Neuroinflammation | Volume 3 | Issue 2 | February 15, 2016