Primer sequence and reaction conditions for PCR amplification.

<p>Primer sequence and reaction conditions for PCR amplification.</p

Establishment and Characterization of a Buffalo (<em>Bubalus bubalis</em>) Mammary Epithelial Cell Line

<div><h3>Background</h3><p>The objective of this study was to establish the buffalo mammary epithelial cell line (BuMEC) and characterize its mammary specific functions.</p> <h3>Methodology</h3><p>Buffalo mammary tissue collected from the slaughter house was processed enzymatically to obtain a heterogenous population of cells containing both epithelial and fibroblasts cells. Epithelial cells were purified by selective trypsinization and were grown in a plastic substratum. The purified mammary epithelial cells (MECs) after several passages were characterized for mammary specific functions by immunocytochemistry, RT-PCR and western blot.</p> <h3>Principal Findings</h3><p>The established buffalo mammary epithelial cell line (BuMEC) exhibited epithelial cell characteristics by immunostaining positively with cytokeratin 18 and negatively with vimentin. The BuMEC maintained the characteristics of its functional differentiation by expression of β-casein, κ-casein, butyrophilin and lactoferrin. BuMEC had normal growth properties and maintained diploid chromosome number (2n = 50) before and after cryopreservation. A spontaneously immortalized buffalo mammary epithelial cell line was established after 20 passages and was continuously subcultured for more than 60 passages without senescence.</p> <h3>Conclusions</h3><p>We have established a buffalo mammary epithelial cell line that can be used as a model system for studying mammary gland functions.</p> </div

Immunostaining for casein in BuMECs

<p>A: Fluorecent image of casein producing BuMECs with relatively stronger signal from cells associated with domes (arrow); B: Light microscope image of same field as in A; C: Negative control experiments with rabbit IgG (Isotype control); D: Light microscope image of same field as in C. The secondary antibodies were rabbit anti-mouse FITC conjugated antibody. DAPI was used as nuclear counter stain. Bars 100 µm. Results represent minimum of three independent experiments.</p

Immunostaining for cytoskeletal markers in BuMECs.

<p>A: Fluorescent image of BuMECs stained for Cytokeratin 18 showing intermediate filaments; B: Fluorescent image of BuMECs stained for Vimentin; C: Negative control with primary antibody replaced with a normal mouse IgG (Isotype control). The secondary antibodies were goat anti-mouse FITC conjugated antibody. Propidium iodide was used as a nuclear counter stain. Bars 100 µm. Results represent images from three independent experiments.</p

RT-PCR analysis for β-casein (I), κ-casein (II), Butyrophilin (III) and Lactoferrin (IV) in BuMECs.

<p>L: 100 bp ladder; A: BuMECs; B: Mammary Tissue (Positive control) and C: Skin fibroblasts (Negative control); Loading control represents the house keeping gene Glyceraldehyde 3–phosphate dehydrogenase (GAPDH). Results representative of minimum three independent experiments.</p

Analysis of casein expression in BuMECs.

<p><b>A:</b>Western blot analysis for casein in BuMEC lysates, M-Protein MW standard, A-Buffalo milk, B-Mammary tissue lysate (Positive control), C-BuMEC lysate, D-Skin fibroblasts lysate (negative control) ACTIN- Loading control. <b>B:</b> Western Blot analysis for casein in BuMEC conditioned media, M–Protein MW standard, A-Buffalo milk, B-Concentrated growth medium (negative control), C-BuMEC conditioned medium, D-Conditioned media from Skin fibroblasts a; α-casein, b; β-casein, c; κ-casein in A and B.</p

Analysis of cell Senescence–associated β-galactosidase (SA-β-gal) activity in BuMECs.

<p>A: SA- β-gal staining in early passage (10) BuMECs (×400); B: SA β-gal staining in late passage (60) BuMECs; Staining is evident from BuMEC with normal morphology and cells with vacuoles (arrow) (×400); Results represents images from three independent experiments.</p

Growth curves for BuMECs at early passage (10), late passage (60) and after revival from cryopreservation (passage 25).

<p>Results are means ±SD of three independent experiments.</p

Development of papillate structures in BuMECs on the plastic substratum.

<p>A: BuMECs from passage 6 forming papillate (arrow) structure after 15 days of growth on plastic substratum (×100); B: BuMECs from passage 8 forming papillate (arrow) structures after 15 days of growth on plastic substratum (×100). Papillate structures represent a small nipple-like projection above the plastic substratum indicating differentiation characteristics of BuMECs.</p

Insulin and Hydrocortisone increases dome formation in BuMECs.

<p>A: BuMECs grown in the absence of insulin and hydrocortisone (×200); B: BuMECs grown in the presence of insulin and hydrocortisone showing increased formation of domes (arrow) (×200). Results represent three independent experiments.</p