Six- to 8-wk-old male and female C57BL/6J mice were purchased from the Jackson Laboratories (Bar Harbor, ME) and allowed to acclimate for one week prior to experimental use. Mice were bred in house and two-week-old neonatal mice were used in all experimental protocols. All procedures utilizing animals have been reviewed and approved by the LSUHSC-S Animal Care and Use Committee.
The following monoclonal antibodies (mAbs) were purchased from either eBioScience (San Diego, CA) or BD-PharMingen ( San Diego, CA): APC-conjugated anti-B220, PE-conjugated anti-B220 (Clone RA3-6B2), APC-conjugated anti-Gr-1 (Clone RB6-8C5), APC-conjugated anti-CD19 (Clone 6D5), APC-conjugated anti-CD3 (Clone17A2), APC-conjugated anti-CD4 (Clone GK1.5), APC-conjugated anti-CD8 (Clone 53-6.7), APC-conjugated anti-Mac-1, FITC-conjugated anti-Mac-1 (Clone M1/70), APC-conjugated anti-Ter-119 (clone Ter-119), APC-conjugated anti-Sca-1 (Ly-6A/E, Clone D7), APC-Cy7-conjugated anti-CD117 (c-Kit, Clone 2B8), and FITC-conjugated anti-CD24 (HSA, clone M1/69). Two isotype controls were purchased from eBioScience: FITC-conjugated Rat IgG2b κ, control for rat IgG2b antibodies, and PE-conjugated Rat IgG2a κ, control for rat IgG2a antibodies. Two other isotype controls, APC-Cy7-conjugated Rat IgG2a κ and APC-conjugated Rat IgG 2b κ, were purchased from CalTag (Burlingame, CA). PE-conjugated anti-CD43 (Clone S7) and APC-Cy7-conjugated anti-CD19 (Clone 1D3) were purchased from PharMingen (San Diego, CA). Anti-Fc receptor mAb (anti-FcR, clone 2.4G2) was produced in house from cells purchased from American Type Culture Collection (Rockville, MD).
Recombinant mouse Stem Cell Factor (SCF), mouse Flt-3/Flk-2 Ligand (FL), mouse interleukin-3 (IL-3), mouse granulocyte macrophage colony stimulating factor (GM-CSF), and mouse interleukin-7 (IL-7) were purchased from R&D Systems (Minneapolis, MN).
Flow Cytometry and Cell Sorting Strategy
ONP cells were sorted from bone marrow of two week-old C57BL/6J mice as previously described [17
]. Cell viability was measured by staining with Trypan Blue (Sigma) and cell samples with viability of more than 98% were used for cell sorting. Dead cells were excluded from analysis by forward angle and 90 degree light scatter gating. Analyses of cells were performed on a FACS Vantage SE Turbo Sorter flow cytometer/sorter (Becton Dickinson, San Jose, CA) or a FacsCalibur cytometer (Becton Dickinson), made available through the University Research Core Facility at LSU Health Sciences Center (Shreveport, LA). A lineage cocktail was prepared from a mixture of APC-conjugated antibodies specific for Mac-1 (CD-11b), Gr-1, Ter-119, B220, CD19, CD3, CD4, CD8, and Sca-1. Lin−
stained cells were sorted on FACS Vantage SE Turbo Sorter. After ONP cells were sorted, post-sort analysis was performed and showed the average of over 95% of purity (). Cultured cells were stained using the appropriate mAbs and isotype-matched antibodies were used as negative controls. All samples were treated with an unlabeled anti-FcR to prevent inappropriate binding of antibodies. Data analysis was accomplished using FlowJo software (TreeStar, San Carlos, CA).
Sort strategy for isolation of the Lin−HSAloCD43loc-Kit+ (ONP) cells from bone marrow of 14-day-old mice
A two-step culture strategy was used to grow the hematopoietic progenitors. Sorted cells were placed into 24 well culture plates (Costar, Cambridge, MA) containing complete IMDM supplemented with 10% fetal bovine serum, 10ng/ml recombinant mouse SCF, 10ng/ml recombinant mouse FL, and 10ng/ml recombinant mouse IL-3. After 3 days, cell cultures were washed and divided into two wells under two different culture conditions. Media in the first set of cultures contained SCF, FL, IL-3, and IL-7 (10ng/ml) to facilitate cell differentiation to B-lineage [19
] and the second set of cultures contained SCF, FL, IL-3, and GM-CSF (10ng/ml) to facilitate cell differentiation along the myeloid pathway [20
]. Cells were then grown for an additional 9 days and were fed every 2–3 days.
To determine the effects of ethanol on the growth and differentiation of the ONP cells, various concentrations of ethanol were added to the growth medium on Day 0. At the time of feeding, ethanol at the appropriate concentration was added to the culture medium used for feeding. Initial analyses were done to determine the stability of the ethanol concentration in the culture medium using a NAD-alcohol-dehydrogenase assay. The concentration of ethanol in the culture medium did not change significantly over a 14 day period (data not shown), thus, with periodic feeding (culture medium changed every 48 hours) the cells were exposed to a constant concentration of ethanol.
Cell Proliferation Assay
To measure the cell viability and proliferation in the presence of ethanol, ONP cells were sorted and cultured with varying concentrations of ethanol (0mM, 25mM, 50mM, 100mM, and 200mM). Cells were cultured as described above. During the culture, cells were harvested at the different time points and prepared for the proliferation assay using CellTiter 96 Aqueous Non-radioactive cell Proliferation Assay kit (Promega, Madison, WI).
Quantitative Real-time RT-PCR Analysis of Transcription Factors (Pax5, EBF, and PU.1), and the Cytokine Receptor
RNA was prepared from ONP cells using an RNA extraction kit (Qiagen Inc., Valencia, CA). cDNA was generated with the ThermoScript RT-PCR kit (GibcoBRL). Quantitative real-time PCR was performed using the Applied Biosystems Model 7700 sequence detection system (Foster City, CA). Primers and probes were designed using Primer Express Software (Applied Biosystems) and were as previously described [17
]. mRNA for the transcription factors Pax5, EBF, and PU.1 and cytokine receptor IL-7Rα were quantitatively measured using GAPDH as an internal control. The relative expression of EBF, Pax5, PU.1, and IL-7Rα versus GAPDH in ONP cells was calculated by determining the ΔCt value for each sample. The ΔCt value = (threshold cycle for the transcriptional factor - the threshold cycle for GAPDH); i.e. the number of additional cycles at which the sample reached the threshold value after the GAPDH threshold value. Therefore, the amount of input RNA for a given sample relative to GAPDH is given by 2−ΔCt
Each experiment was repeated at least three times and Student t test and ANOVA of mean values from each experiment were used in comparison of differences in cell populations between the ethanol and normal control groups and analyzed by a statistical software package (Instat, Graphpad Software, San Diego, CA). Differences between means were considered significant when p<0.05.