Table 1.

Immunostaining of microglia to mGlu receptors and activation markers

Percentage of total number of cells in control microglial cultures staining with markers
Isolectin B4	99.05  ± 0.95
OX-42	98.97  ± 1.22 (p = 0.9600)
mGlu4a	92.83  ± 1.84 (p = 0.0305)
mGlu6	97.11  ± 0.19 (p = 0.1896)
mGlu7	0.87  ± 0.54 (p < 0.0001)
mGlu8	91.84  ± 3.78 (p = 0.2280)
Percentage of total number of cells staining with ED1
Control	2.93  ± 0.69
l-AP-4	36.60  ± 0.01
RS-PPG	38.65  ± 3.89
CGA	77.80  ± 4.70
FACS data for the percentage of cells staining with ED1
	% Positive events	Mean fluorescence intensity
Control	16.66	9.22
l-AP-4	30.39	9.69
RS-PPG	32.05	10.58
CGA	50.10	29.87
LPS	69.29	24.46

Top, Percentage of cells staining with isolectin B4, OX-42, and antibodies to mGlu4a, mGlu6, mGlu7, and mGlu8 receptors. Primary cultured microglia were fixed after 1 DIV and stained withGriffonia simplicifolia isolectin B4 (1:20) or OX-42 (1:1000) and mGlu4a, mGlu6, mGlu7, or mGlu8 antibodies (all at 1:250) and counterstained with DAPI (1:1000) for total cell number. Cells were counted in three separate fields on four coverslips per condition and expressed as a mean ± SEM percentage of total cell number (DAPI-positive cells). p values are given for comparison by ANOVA, followed by pairwise analysis by Student's t test with isolectin B4 staining.

Middle, Percentage of cells staining with ED1, a microglial marker for activation. Primary microglial cultures were treated for 24 hr with 100 μml-AP-4, 100 μmRS-PPG, or 50 nm CGA or solvent only (controls). Cells were fixed and stained with ED1 (1:500) and counterstained with hematoxylin. Cells were counted in three separate fields on four coverslips per condition, and the number of ED1-positive cells are expressed as a percentage of total cell number (hematoxylin-positive cells). Mean ± SEM of three separate fields on four coverslips per condition.

Bottom, FACS analysis for the percentage of cells staining with ED1 after exposure of microglial cultures to mGlu receptor agonists and activators. Primary cultured microglia were treated for 24 hr with 100 μml-AP-4, 100 μmRS-PPG, 50 nm CGA, 1 μg/ml LPS, or solvent only (controls). Cells were fixed and incubated with ED1:FITC (0.1 mg/ml) and subjected to FACS analysis. Ten thousand cells per sample were counted, and results are expressed as the percentage of cells fluorescent above a no-antibody control (% Positive events) and the logarithmic average of these values also obtained (Mean fluorescence intensity).