MATERIALS AND METHODS

4.2.1. TARGET PREPARATION FOR MICROARRAY HYBRIDISATION

4.2.1.1. Primary cell culture and ionomycin treatment

The flow diagram in Figure 4:1 shows the methods used in microaiTay gene expression analysis in this study. In order to obtain skeletal muscle myocytes, a 4-weeks-old Large White cross pig from a local farm was used. Longissimus dorsi muscles were removed from the pig, and washed in PBS DULBECCO’S [without (w/o)/lacking calcium, magnesium and sodium bicarbonate] (Gibco'^'^, Invitrogen Corporation) in a 50 ml Falcon tube (Becton Dickinson). The muscle tissues were transferred to a 140 mm sterile Sterilin petri dish (Bibby Sterilin Ltd) in a BioMAT class II microbiological safety cabinet (Medical Air Technology Ltd), and finely dissected using sterile Swann-Morton® disposable scalpels. About 5 ml of the finely minced muscle tissue was transferred to a new 50 ml Falcon tube. Filter-sterilised dispase 1.4 U/ml (Sigma) in PBS DULBECCO’S was added to the minced muscle to obtain a final volume of 50 ml. The mixture of the minced muscle and dispase was incubated at 37 °C for two hours, during which the mixture was shaken fairly vigorously every few minutes. Following incubation, the mixture was centrifuged at lOOx g at room temperature for 5 minutes to pellet tissue debris. Debris was removed, and the supernatant was transferred to a new 50 ml Falcon tube using a sterile disposable plastic pipette. Dulbecco's modified Eagle's medium (DMEM) with glutamax-1 (with sodium pyruvate, 4500 mg/1 glucose and pyridoxine) (GIBCO™, Invitrogen Corporation), supplemented with 15% [volume/volume (v/v)] heat inactivated foetal bovine serum (Sigma- Aldrich), 50 mg/L penicillin (Invitrogen), 100 mg/L streptomycin (Invitrogen) and 10 pg/ml ciproxin (Bayer), was added to the supernatant to obtain a final volume of 50 ml, followed by centrifugation at 400x g at room temperature for 5 minutes to pellet cells. Following centrifugation, the supernatant was decanted, and the cell pellet was washed with 20 ml DMEM with supplements. The suspension was centrifuged at 400x g for 5 minutes at room temperature to re-pellet cells. Following centrifugation, the supernatant was decanted and the cell pellet was re-suspended in 10 ml DMEM with supplements. The re-suspended cells were filtered using sterile lOO-pm nylon cell strainers (Becton Dickinson) into a new 50 ml Falcon tube.

Figure 4:1 Flow diagram showing methods used for microarray gene expression analysis in this study

The target cDNA mixture

was hybridised to eight in-house constructed porcine skeletal mnscle cDNA microarray slides Differentiated myocytes

treated with ionomycin Differentiated myocytes without any treatment

Calcinuerin phosphatase assay

Preparation of target cDNA mixture

cDNA synthesis and labelling cDNA with fluorescent cyanine dye cDNA synthesis and labelling

cDNA with fluorescent cyanine dye

Culturing skeletal muscle satellite cells

Isolation of mRNA from the harvested cell pellets Isolation of mRNA from the

harvested cell pellets

Isolation of longissimus dorsi muscle from a 4-6 weeks-old young pig

Fifty of sterile Costar'' 162 cm^ non-pyro genic, polystyrene cell culture flasks (Costar ", Corning Incorporated) were used for the primary cell culture. One microlitre of the filtrate was transfeiTed into each of the fifty cell culture flasks. Fifteen microlitres of DMEM, supplemented with 15% (v/v) heat inactivated foetal bovine serum (Sigma-Aldrich), 50 mg/L penicillin (Invitrogen), 100 mg/L streptomycin (Invitrogen) and 10 pg/ml ciproxin (Bayer), was added to the filtrate in each flask. Some cells were plated onto ten chamber glass slides for fixation and morphological analysis and desmin detection. The satellite cells cultured in each 162 cm^ flask was maintained for 48 hours at 37 °C with 5% CO2 in air in a humidified HERA cell incubator (Heraeus). During the culturing process, cells were examined using a Leitz “Labovert FS” trinocular inverted microscope (Leitz). Following incubation, the medium in each flask was discarded. One millilitre of Trypsin-EDTA (Ix) in HESS w/o Ca and Mg w/EDTA.4NA (GIBCO™, hivitrogen Coiporation) was added to cells in each flask, incubated at 37“C with 5% CO2 in air in the humidified cell incubator for 5 minutes, and then transferred into a new 50 ml Falcon tube, followed by centrifugation at 120Qx g for 5minutes. Cell pellets were re-suspended with 40 ml fresh DMEM with supplements, and transferred into new 162 cm^ flasks. The cells were maintained at 37°C with 5% CO2 in air in the humidified cell incubator for another 120 hours (5 days), during which the medium was replaced every two days. Following five days of incubation, the cells in each flask became about 80-90% confluent. DMEM containing foetal bovine serum in each flask was discarded, and 25 ml of DMEM containing 2% (v/v) heat inactivated horse serum (Sigma-Aldrich), 50 mg/L penicillin, 100 mg/L streptomycin and 10 pg/ml ciproxin were then added to cells. Cells were maintained at 37°C with 5% CO2 in air in the humidified cell incubator for 96 hours (4 days) for differentiation, during which the medium was replaced every two days (Kubis et al., 1997).

After 96 hours of incubation, the medium was removed, and the cells were maintained in 25 ml fresh DMEM with supplements in the absence or presence of ionomycin calcium salt (Sigma- Aldrich). Ionomycin calcium salt was initially dissolved in dimethyl sulphoxide (DMSO) and sterilised using a sterile 0.25 pm filter (Nalgene) to give 1 mM stock solutions. Fifty microlitres of ImM ionomycin stock solution were diluted with 25 ml DMEM (with supplements) to obtain a final concentration of 2 pM. DMEM (with supplements) containing 2 pM ionomycin calcium salt was added to the myocytes cultured in twenty-five 162 cm^ flaks. The myocytes cultured in the remaining twenty-five 162 cm^ flasks were treated with a vehicle (DMSO) in the absence of ionomycin treatment, serving as a control. DMSO alone had no effect on cultured myocytes at

the concentrations used (0.001-0.05 % v/v) (Scott et al., 1997; Sun et al., 1998). Both cell groups were maintained at 37°C with 5% CO2 in air in the humidified cell incubator for 48 hours prior to harvest.

4.2.1.2. Detection of desmin expression in the cultured cells

4.2.1.2.1. Cell culture on glass slides and methanol/acetone fixation for adherent cells

Skeletal muscle satellite cells were also cultured on Lab-Tek® II chamber glass slides (Nalge Nunc International). Materials and methods used for the cell culture on chamber glass slides were the same as that described for the cell culture in 162 cm^ flasks (section 4.2.1.1. of this chapter). Following cell culture, the medium was discarded, the glass slides with cells adherent on one side of them were immersed in 1:1 ice-cold methanol:acetone incubated at -20°C in a freezer for 10 minutes. The glass slides were then removed fr'om the -20°C freezer, air-dried at room temperature and then subjected to immunohistochemical staining for desmin.

4.2.1.2.2. Immunohistochemical staining o f the cultured cells

The glass slides with fixed cells on one side were placed in a metal rack and immersed briefly in ddH20. The glass slides were then placed for 30 minutes in 100 ml methanol containing 0.5% H2O2, and washed briefly in Tris-buffered Tween. About 1 ml of 1% rabbit serum solution was applied to the cells, and then incubated at room temperature for 30 minutes. Following incubation, 1:500 diluted monoclonal mouse anti-desmin antibody (mouse Ig concentration was 160 mg/L; total protein concentration was 26 g/L) (DAKO), which reacts with inteimediate filament protein desmin in skeletal muscle myocytes, was applied to the cells on the glass slides. The cells were incubated at room temperature for 2 hours and then washed with Tris-buffered Tween for three times with 5 minutes for each time. About 1 ml of 1:200 diluted rabbit anti­ mouse biotinylated antibody was applied to the cells on the glass slides. The cells were incubated for 45 minutes and then washed with Tris-buffered Tween for tlnee times with 5 minutes each time. About 1 ml of SteptAB Complex/HRP (Dako) was applied to the cells on the glass slides. The cells were incubated for one hour and then washed with Tris-buffered Tween for three times, each time for 5 minutes. A solution of 3,3 diaminobenzidine was applied to the cells on the glass slides, which were examined under the light microscope. The cells were then washed in

ddH20 to terminate the reaction. The cells on glass slides were placed in Gills Haematoxylin for 30 seconds, then washed in ddH20. The cells on glass slides were immersed briefly (about 3 dips) in 1% acid alcohol, and then washed in ddH20. The cells on glass slides were immersed in STWS for staining nuclear, and then washed in ddH20. The glass slides were placed in methylated spirits, then in absolute alcohol, and finally in citroclear solution. Permanent mountant was then applied to the cells on glass slides.