Antibiotic antimycotic solution

Animals-Wistar rats (Charles River Laboratories, Arbresle, France) were kept in a temperature-controlled room (21 Ϯ 1 °C) under an established photoperiod (lights on 07.00 -19.00 h) with free access to food and tap water. Animal manipulations were performed according to the recommendations of the French Ethics Committee and under the supervision of authorized investigators.
Reagents-Multiple-associated peptide (MAP)-C3a and MAP-C5a are eight peptidic monomers corresponding to the last 13 amino acids in the C-terminal region of the anaphylatoxins attached to a polylysine comb (a gift of Dr. Ischenko). These peptides were previously used and were shown to exhibit the same activity as recombinant or natural anaphylatoxins (41) (42) (43) . C5aR antagonist (44) was synthesized by Dr J. Leprince (INSERM U413). Anti-mouse C3aR and anti-rat C5aR were obtained by immunization of rabbits with recombinant fusion proteins consisting of glutathione S-transferase fused to the region 161-333 of the large extracellular loop of mouse C3aR and to the 29-amino acid peptide corresponding to the 8 -36 sequence of the N-terminal region of rat C5aR (according to EMBL library index accession numbers NM_009779 and Y09613, respectively).
Cell Culture-Granule cell suspensions were prepared from cerebelli of 8-day-old Wistar rats, as described previously (45) . Dispersed cells were seeded in multiwell plates or dishes (Falcon, BD Biosciences) coated with 10 M poly-L-lysine (Sigma) at a density of 2.5 ϫ 10 5 cells per cm 2 . Granule neurons were plated in a medium consisting of 75% Dulbecco's modified Eagle's medium (Invitrogen) and 25% Ham's F-12 medium (Sigma) containing 2 mM glutamine, 1 mM sodium pyruvate, 1% antibiotic-antimycotic solution (BioWhittaker, Verviers, Belgium) supplemented with 25 mM KCl and 10% fetal calf serum (SϩK25). Proliferation of non-neuronal cells was blocked by the addition of 10 M cytosine ␤-D-arabinofuranoside (Sigma) on day 1 in vitro (DIV 1). Cultures prepared by this method were enriched in granule neurons by more than 95%; the cell population was negative for glial fibrillary acidic protein (Sigma), OX 42 (ATCC, Manassas, VA), and calbindin D28K (Sigma) staining (data not shown). Cells were grown at 37 °C in a humidified incubator with an atmosphere of 5% CO 2 .
RNA Isolation and Real-time Quantitative Reverse Transcription (RT)-PCR-Total RNAs were extracted from cultured granule cells by the guanidinium isothiocyanate method followed by ultracentrifugation onto a CsCl cushion as previously described (46) . Total RNAs (2 g) were incubated at 70 °C for 10 min, and RT was carried out in a thermocycler (Hybaid, Cera-Labo, Ecquevilly, France) at 37 °C for 1 h with 5 mM dithiothreitol, 1 mM dNTPs, 500 pmol of random hexamer primers pdN6 (Amersham Biosciences), 120 units of RNasin (Promega, Charbonnie `res, France), and 400 units of Moloney murine leukemia virus (M-MLV) RT (Promega) in the reaction buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl 2 , and 5 mM dithiothreitol). The absence of genomic contaminant was checked routinely by RT-PCR in negative control samples in which either the RNA samples were replaced with sterile water or the Moloney murine leukemia virus RT was omitted.
PCR was carried out by using the LightCycler FastStart DNA Master SYBR Green I kit (Roche Applied Science), which is specially adapted for the LightCycler instrument (Roche Applied Science), and the PCR reaction was performed in a final volume of 20 l in the LightCycler glass capillaries according to the manufacturer's recommendations. The primers used in our study were: RC3a forward (GAC CTA CAC TCA GGG C), RC3a reverse (ATG ACG GAC GGG ATA AG), RC5a forward (ATG CCT GCA GAT GGC GTT TA), and RC5a reverse (CAG AAA CCA AAT GGC GTT GAC). The housekeeping gene primers were glyceraldehyde-3-phosphate dehydrogenase forward (TGC CAT CAA CGA CCC CTT CA) and glyceraldehyde-3-phosphate dehydrogenase reverse (TGA CCT TGC CCA CAG CCT TG). Primers were chosen according to their cDNA sequences reported in the EMBL data library index accession numbers M33197 for glyceraldehyde-3-phosphate dehydrogenase, NM_032060 for C3aR, and Y09613 for C5aR. Southern Blot Analysis-RT-PCR products (5 l) were loaded onto a 1% agarose gel and separated by electrophoresis. The gel was treated for 10 min in 0.15 M HCl and neutralized for 30 min in 0.4 M NaOH. Southern blotting was performed by capillary transfer for 16 h in 0.4 M NaOH onto Nylon Plus membranes (Amersham Biosciences). The blot was neutralized for 10 min in 2ϫ SSPE (0.3 M NaCl, 17 mM Na 2 HPO 4 , and 50 mM EDTA). Rat C5aR and mouse C3aR cDNA probes were labeled by using Rediprime Kit (Amersham Biosciences) with [ 32 P]dCTP (Amersham Biosciences) at a specific activity of 2 ϫ 10 9 cpm/g. Membranes for C5aR were pre-hybridized in homologous conditions at 42 °C for 4 h in a solution containing 50% formamide, 5ϫ SSPE, 1% SDS, 5ϫ Denhardt's, 5% dextran sulfate, and 100 g/ml herring sperm DNA, whereas membranes for C3aR were pre-hybridized in heterologous conditions in a solution containing 20% formamide, 4ϫ SSPE, 0.05 M Tris-HCl, pH 7.5, 1 M NaCl, 0.1% SDS, 10ϫ Denhardt's, and 100 g/ml herring sperm DNA. Hybridization was performed at 42 °C for 16 h in the same solutions supplemented with 10 8 cpm of labeled probe. The membranes were washed briefly 3 times at room temperature in 2 ϫ SSPE, 0.1% SDS, for 1 h at 68 °C for C5aR and 60 °C for C3aR in 2ϫ SSPE, 0.1% SDS, and for 1 h at 68 °C for C5aR and 60 °C for C3aR in 1ϫ SSPE, 1% SDS and exposed for 4 h at room temperature onto X-Omat film (Eastman Kodak Co.).
Flow Cytometry Analysis-Neurons were harvested from cultures by incubation in phosphate-buffered saline (PBS) containing 10 mM EDTA. Cells were washed and resuspended in PBS containing 1% bovine serum albumin (BSA) and incubated with 10 g/ml non-immune mouse IgG for 15 min. After washing, cells were incubated with 2 g/ml anti-C3aR or anti-C5aR IgG or anti-␥-aminobutyric acid, type A receptor ␣6 subunit (Chemicon, Euromedex, Souffelweyersheim, France) diluted 1:100 in PBS containing 1% BSA at 4 °C for 30 min. After washing, cells were incubated at 4 °C for 30 min with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Jackson Immuno Research, Interchim, Montluc ¸on, France) diluted at 1:200 and measured in a FL-1 channel (530 Ϯ 15 nm bandpass filter). For two-color flow cytometric analysis, cells were incubated in PBS containing 1% BSA and 0.1% saponin for 30 min with biotinylated anti-C3aR or biotinylated anti-C5aR (2 g/ml) and monoclonal anti-neurofilament 68 clone NR4 (Sigma) diluted 1:100. After washing, cells were incubated for 30 min with streptavidin cyanine 5 (measured in FL-4 channel, 661 Ϯ 8 nm bandpass filter) diluted 1:500 and with fluorescein isothiocyanateconjugated goat anti-mouse IgG (FL-1) diluted 1:200. Cells were washed before analysis on a FACScalibur flow cytometer (BD Biosciences) operating with the Cell Quest TM software. Dead cells and debris were excluded from the analysis by gating living neurons from size/structure density plots. Data were displayed on a logarithmic scale with increasing fluorescence intensity. Each histogram plot was recorded for at least 10,000 gated events.
Immunocytochemistry-Granule cells were fixed with 1% formaldehyde in PBS at room temperature for 20 min. Fixed cells were incubated 1 h at room temperature with 2 g/ml anti-C3aR or anti-C5aR or with anti-neurofilament 68 diluted 1:100 in PBS containing 1% BSA. After several rinses in PBS, cells were incubated at room temperature for 2 h with peroxidase-conjugated Affinipure goat anti-rabbit or anti-mouse IgG (Jackson Immuno Research) diluted 1:1000 in PBS containing 1% bovine serum albumin. Cells were washed twice with PBS. The immunoreactivity was revealed by adding 0.5 mg/ml diaminobenzidine (Sigma) and 0.015% H 2 O 2 in PBS. The reaction was stopped by removing the diaminobenzidine solution and by rinsing in a large volume of water. The immunolabeling was analyzed using a computer-assisted microscope AxioVert (Zeiss, Le Pecq, France).
Immunohistochemistry-Rats were deeply anesthetized with chloral hydrate (400 mg/kg intraperitoneal) and transcardially perfused and fixed with 4% paraformaldehyde in PBS. The cerebellum was removed, post-fixed for 12 h in 4% paraformaldehyde, and placed in 15 and 30% sucrose until they sank. Tissue sections (10-m thick) were cut with a cryostat (Leica, Rueil-Malmaison, France). For immunohistochemical detection, endogenous peroxidase was blocked by incubating sections in 3% H 2 O 2 in methanol at room temperature for 10 min. Then fixed tissues were incubated at 4 °C overnight with 2 g/ml anti-C3aR or anti-C5aR or anti-neurofilament 68 in PBS containing 0.3% Triton X-100 and 1% BSA. After several rinses in PBS, the tissues were incubated at room temperature for 2 h with peroxidase-conjugated Affinipure goat anti-rabbit or anti-mouse IgG diluted 1:1000. After several rinses in PBS, the sections were incubated at room temperature for 2 h with the peroxidase-anti peroxidase complex (Sigma) diluted 1:200 in PBS. The sections were washed twice with PBS and rinsed with 0.1 M Tris-HCl buffer. The immunoreactivity was revealed by using a 3,3Ј-diaminobenzidine substrate kit containing 0.02% H 2 O 2 (Sigma) and nickel ammonium sulfate. The reaction was stopped by removing the diaminobenzidine solution and by rinsing with a large volume of water. The specificity of immunolabeling was checked by omitting the primary antibody and by replacing the antiserum with pre-immune serum. Microphotographs were acquired on a computer-assisted image analyzer (Biocom 2000, Les Ulis, France).
Immunoprecipitation and Western Blot-Total cellular proteins were extracted from DIV 4 neurons in lysis buffer containing 0.15 M NaCl, 0.05 M EDTA, 4% CHAPS, and 0.2% of the protease inhibitor mixture (0.5 M EDTA, 2.5 M benzamidine, 2.18 mM pepstatin A, 11.7 M leupeptin) for C3aR detection and 1% Triton X-100, 25 mM Tris-HCl, 5 mM EDTA, 250 mM NaCl, 10% glycerol and 0.2% of the protease inhibitor mixture for C5aR, Bax, and Bcl-2 detections. Spleen or cerebellum of 12-day-old rat were lyophilized, and the dry powders were resuspended in the corresponding lysis buffer (10 ml per g of lyophilized tissue) and incubated overnight at 4 °C. These extracts were centrifuged (15,000 ϫ g, 4 °C, 15 min), and the supernatant was immunoprecipitated before Western blotting to enrich extracts in C3aR or C5aR proteins. Freshly made cell lysates were incubated with anti-C3aR or anti-C5aR at 4 °C for 30 min. The lysates were then incubated with protein A-Sepharose at 4 °C for 1 h under gentle rotation. After centrifugation at 10,000 g for 5 min, the supernatant was discarded. Immunoprecipitates were washed 5 times with PBS. The resulting pellet was solubilized in Laemmli sample buffer, reduced with the addition of mercaptoethanol (47) , and boiled for 5 min. The immunoprecipitate proteins were separated by 10% SDS-PAGE on 12% gels and electrotransferred to polyvinylidene difluoride membranes (Immobilon-P Millipore, Saint-Quentin en Yvelines, France). Nonspecific sites were blocked with 5% nonfat milk at room temperature for 30 min, and the membrane was incubated overnight at 4 °C with biotinylated anti-C3aR, biotinylated anti-C5aR (2 g/ml), anti-Bax (1:200, sc-493, Santa Cruz Biotechnology, CA), or anti-Bcl-2 (1:200, sc-492, Santa Cruz Biotechnology) with rocking. After washing, bands were visualized by incubation with streptavidin-horseradish peroxidase or conjugated Affinipure goat anti-rabbit IgG (1:1000) for 2 h followed by chemiluminescence detection (ECL detection kit, Amersham Biosciences).
Measurement of Calcium Flux-Cells were loaded with 4 M fluo-4AM (Molecular Probes, Interchim) in HBK buffer (120 mM NaCl, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 6 mM glucose, 10 mM HEPES, pH 7.4) for 30 min at 37 °C. After washing twice with HBK buffer, fluorescence intensity was measured with a FL600 microplate reader (Bio-Tek Instruments, Winooski, VT) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm every 15 s for 6 min.
Apoptosis Induction-Cerebellar granule neurons were cultured for 4 days in SϩK25. The cells were washed twice and cultured in serum-free medium containing low (5 mM) potassium concentration (S-K5). Under these conditions cerebellar granule neurons degenerate and die by apoptotic cell death within 8 -24 h (37) (38) (39) .
Cell Viability-Cells were incubated with 1.3 M calcein AM (Molecular Probes) in PBS for 15 min. After washing twice with PBS, fluorescence intensity was measured with a FL600 microplate reader (Bio-Tek Instruments) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Pilot experiments have shown that the fluorescence intensity is proportional to the number of viable cells (in the range 5 ϫ 10 4 to 1 ϫ 10 6 cells/ml).
DNA Fragmentation-Internucleosomal DNA cleavage was assessed by conventional gel electrophoresis after extraction of nuclear DNA by using the Wizard Plus Minipreps DNA purification system (Promega). Granule neurons were incubated for 10 min at room temperature in a lysis buffer consisting of 50 mM Tris-HCl, 10 mM EDTA, 1% Triton X-100, and 50 g/ml RNase A. After centrifugation at 14,000 ϫ g for 15 min, the cleared lysates were mixed with the Wizard resin and transferred into a vacuum manifold column. Three washes with the columnwash solution were performed, and DNA fragments were eluted with 50 l of water at 60 °C. DNA ladders were visualized on a 1.5% agarose gel.
Measurement of Caspase Activity-For measurement of caspase-3 activity, cultured cells were washed twice with PBS at 37 °C, resuspended in Dulbecco's modified Eagle's medium (100 l), and treated with the fluorometric caspase-3 assay system (Promega). In brief, caspase substrate and homogeneous caspase buffer (100 l) were mixed and added to the cells (100 l) in 96-well plates. The fluorescence intensity was measured with a Bio-Tek FL600 microplate reader (Bio-Tek Instruments) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm every 15 min for 3 h. For measurement of caspase-9 activity cultured cells were washed twice with PBS at 37 °C and resuspended in PBS at 4 °C. Cells were harvested by centrifugation (350 ϫ g, 4 °C, 9 min) and treated with the fluorometric caspase-9 assay systems (R&D Systems, Minneapolis, MN). In brief, the cell pellet was resuspended in 95 l of hypotonic cell lysis buffer and incubated with 95 l of reaction buffer and caspase-9 substrate in 96-well plates. Fluorescence intensity was measured at an excitation wavelength of 360 nm and an emission wavelength of 530 nm every 15 min for 3 h.
Measurement of Mitochondrial Activity-Cultured cells were incubated in the presence of the JC-1 probe (7.5 g/ml; Molecular Probes) at 37 °C for 15 min and then washed with PBS. In viable granule neurons, the intact membrane potential allows the lipophilic dye JC-1 to enter into the mitochondria, where it accumulates and aggregates, producing an intense orange signal. In apoptotic cells, the mitochondrial membrane potential collapses so that the monomeric JC-1 probe remains cytosolic and stains the cell in green. Fluorescence intensity was measured with a Bio-Tek FL600 microplate reader and expressed as a ratio of the emission at 590 nm (orange) over 530 nm (green).
Statistical Analysis-All values are expressed in means Ϯ S.E. A one-way analysis of variance and a Tukey-Kramer multiple comparisons test were used to determine statistical differences between mean values.