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We used a simple commercial magnetic immunobead method for the preparation of acutely isolated microglial cells from postnatal days 1-3 rat brain. With the exception of a 15 min enzyme incubation, all stages are carried out at 4 degrees C, minimizing the opportunity for changes in gene expression during the isolation to be reflected in changes in accumulated mRNA. The composition of the isolated cells was compared with that of microglial cultures prepared by conventional tissue culture methods, and the purity of microglia was comparable between the two preparations. RT-PCR analysis of several genes related to inflammatory products indicated that the acutely prepared cells were in a less activated condition than the conventionally tissue cultured cells. We examined the pattern of expression of receptors for lysophosphatidic acid (lpa) and sphingosine-1-phosphate (S1P) using quantitative real-time PCR (TaqMan PCR) techniques. mRNA for LPA1, S1P1, S1P2, S1P3 and S1P5 was detected in these preparations, but the levels of the different receptor mRNAs varied according to the state of activation of the cells. mRNA for LPA3 was only detected significantly in cultured cell after lipopolysaccharide (LPS) stimulation, being almost absent in cultured microglia and undetectable in the acutely isolated preparations. The levels of mRNA of LPA1 and S1P receptors was reduced by overnight exposure to S1P, while the same treatment significantly up-regulated the level of LPA3 mRNA.
Tham et al. (Wed,) studied this question.