Agents and materials
The peptides SVPII and SVPIII (used only in AlamarBlue™ assay) were isolated from the venom of Buthus Martti Karsch as described . Recombinant human macrophage colony stimulating factor (rhM-CSF) and recombinant mouse (rm) IL-3 were purchased from PeproTech Co. AlamarBlue™ was purchased from AbD Serotec (Great Britain), and membrane protein isolation kits were from Bio-Rad. An IL-3R antibody was purchased from Abcam Co. Methyl cellulose for CFU assay was from Sigma-Aldrich Co.
The rhM-CSF-dependent cell line M-NFS-60 (CRL-1838™) was purchased from ATCC Co. (USA).
M-NFS-60 cell culture and treatment groups
The M-NFS-60 cell line was cultured in PRMI 1640 culture media supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 U/ml streptomycin, 5.958 g/L HEPES, and 62 μg/L rhM-CSF. Cells were maintained at 37°C under a 5% CO2 atmosphere. The media was changed every other day. Cells were used for experiments in the exponential growth phase. Unirradiated or 60Coγ-irradiated M-NFS-60 cells were treated with PBS (control), SVPII or SVPIII alone, IL-3 alone, or SVP plus IL-3 for various durations.
Special cell culture methods
M-NFS-60 cells were cultured in serum-free media supplemented with 62 μg/L rhM-CSF for 24 h (serum-free) or treated with 3 mg/L SVP II (SVP II) or 10 μg/L IL-3 (IL-3). The control cells were cultured 24 h in normal medium (24 h control). After 24 h, the cell cycle was analyzed by FCM. After cultured in serum-free media plus rhM-CSF for 24 h, the cells were cultured in normal midium (mentioned above) for an additional 72 h (serum-free) or treated with SVPII 3 mg/L or IL-3 10 μg/L in the same media. The control cells were cultured 96 h in normal medium (96 h control). After 96 h, the cell cycle was analyzed by FCM. Serum-free medium will lessen the influence factors on the cell cycle progression.
After irradiation by 60Coγ-ray
M-NFS-60 cells were cultured in PRMI 1640 culture media supplemented with 10% FCS, 100 U/ml penicillin, 100 U/ml streptomycin, 5.958 g/L HEPES, and 15.5 μg/L rhM-CSF (25% of the normal M-CSF dose) for 48 h (irradiated control) or treated with 3 mg/L SVPII or 10 μg/L IL-3 for 48 h. Unirradiated cells were cultured 48 h in the same medium were served as control. After 48 h, the cell cycle was analyzed by FCM.
M-NFS-60 cells were irradiated by 60Coγ-ray at 5 Gy using a Gammacell 3000 Elan installation (Nordion Intern. Inc., Canada). Proliferation and cell cycle progression were then analyzed as described below.
Preparation of mouse BM-MNCs
All animal experiments in this study were approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University. The BALB/C mice were euthanized with CO2 and the femoral bones removed. The femoral bone cavity was washed with low-sugar DMEM medium to harvest bone marrow cells. The cells in DMEM were then slowly added onto the surface of a lymph cell isolation solution and centrifuged at 2000 rpm for 20 min. The annular white layer consisting of monocytes was collected, washed three times in PBS, and resuspended in DMEM at the optimal concentration for each experiment.
AlamarBlue™ cell viability assay
The AlamarBlue assay was used to measure the effect of SVP on the proliferation of non-irradiated and irradiated M-NFS-60 cells cultured in suspension. After irradiation or sham treatment, M-NFS-60 cells were washed three times in PRMI 1640 culture media, and the live cells counted using Trypan Blue vital staining. The cell concentration was adjusted to 5 × 104 cells/mL using PRMI 1640 culture media containing 10% FCS and 62 μg/L rhM-CSF, and aliquoted at 80 μL/well in 96-well plates. After 24 h incubation at 37°C, 10 μL PBS (control), SVP (1, 3, or 4 mg/L), IL-3 (10 μg/L), or SVP + IL-3 was added to each well. Each treatment was performed in triplicate in the same 96-well plate. Following control or drug treatment, 10 μL AlamarBlue™ was added to each well and plates incubated at 37°C for 48 h. Optical density (OD) values were measured and the cell proliferation rate calculated.
Colony forming unit (CFU) assay
A methyl cellulose half-solid colony formation method was adopted to measure the number of bone marrow mononuclear cell CFUs under different treatment conditions. Treated BM-MNCs (1 × 106 cells/mL) were added into methyl cellulose half-solid medium composed of DMEM, 0.8% methyl cellulose, 30% FCS, 2 mmol/L L-glutamine, and the recombinant cytokines (10 μg/L IL-3 and 62 μg/L rhM-CSF). The CFU number was counted under a microscope after 7, 11, and 14 days of incubation at 37°C in a 5% CO2 atmosphere. A mass consisting of more than 50 cells was defined as 1 CFU.
Analysis of the cell cycle using FCM
The M-NFS-60 cells (non-irradiated or irradiated) were treated as described. A 0.5 mL cell suspension (1 − 5 × 106/mL) from each treatment group was combined with 2 ml of cooled 70% ethanol and kept overnight at 4°C, centrifuged at 1000 rpm/min, washed in PBS, and incubated in the dark room at 4°C for 30 min with 50 μL RNAse and 450 μL propidium iodide (PI) staining solution. The proportion of cells in each phase of the cell cycle was then determined by PI staining intensity using FACScalibur flow cytometer (Becton Dickinson).
Detection of IL-3R expression
Cultured M-NFS-60 cells on glass slides were washed twice in PBS, fixed in −20°C pre-cooled 100% methanol for 5 min, dried, and then blocked in 5% BSA solution for 1 h at room temperature or overnight in BSA at 4°C. The blocking solution was removed and anti-IL-3R antibody (Abcam, USA) (1:200) added for 1 h at 37°C or overnight at 4°C. After washing in PBS, an FITC-labeled secondary antibody (F-0382; Sigma) was applied at 1:500 in PBS. Slides were then washed in PBS and sealed in glycerol. The expression of IL-3R was detected by immunofluorescence under a laser scanning confocal microscope (Leica DM1 4000B).
FCM analysis of IL-3R expression
Cells were treated as described, harvested, and the concentration of M-NFS-60 cells adjusted to between 5 × 106 and 1 × 107 cells/mL in PRMI 1640 culture medium. To this cell suspension were added a monoclonal antibody (5–50 μL) and 50 μL inactivated rabbit serum. Cell suspensions were incubated at 4°C for 30 min, washed in PBS, and centrifuged. The supernatants were removed, and the pellets treated with 50 μL of a FITC-conjugated goat anti-mouse antibody, shaken at 4°C for 30 min, washed twice in PBS, centrifuged, and fixed as described in section 3.7.1.
Membrane proteins from the different treatment groups were extracted using a Bio-Rad membrane protein extract kit. Total protein concentrations were measured by the Lowry assay and extracts run on 12% SDS-PAGE gels. Separated proteins were electrotransferred to polyvinyl membranes. Membranes were probed with an IL-3R antibody and visualized using chemiluminescence.
The data are expressed as mean ± SD. SPSS statistical software was used to perform chi-square analysis. P < 0.05 was considered statistically significant.