Materials and Reagents

Bovine cells (The TBL3 cell line was derived from in vitro infection of the spontaneous bovine-B lymphosarcoma cell line, BL3, with Hissar stock of T. annulata)
RPMI 1640 (Gibco)
Fetal calf serum (heat-inactivated)
Penicillin/streptomycin
Fibronectin (Sigma-Aldrich, catalog number: F1141 )
Formaldehyde (Sigma-Aldrich, catalog number: F8775 )
Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
SVF (PAA Laboratories GmbH, catalog number: A15-101 )
BSA (Sigma-Aldrich, catalog number: A2153 )
Mouse monoclonal anti-Ki67 (1:50) (Abcam, catalog number: ab10913-1 )
Cy2 AffinyPure anti-mouse IgG (1:5,000) (Jackson ImmunoResearch Laboratories, catalog number: 715-225-150 )
Tween 20 (Biosolve, catalog number: 2045 2335 )
ProLong Gold Antifade Reagent with Dapi (Life Technologies, Invitrogen™, catalog number: P-36931 )
DAPI

Equipment

Slides (Knittel Glass, catalog number: KN00010025787 )
37 °C, 5% CO₂ cell culture incubator
24 wells plate
Fluorescent microscope (Leica Microsystems, model: Inverted 6000 )

Procedure

Bovine cells were cultured in RPMI 1640, supplemented with 10% heat-inactivated Fetal calf serum, 4 mM L-Glutamine, 25 mM HEPES, 10 μM β-mercaptoethanol and 100 μg/ml penicillin/streptomycin in a humidified 5% CO₂ atmosphere at 37 °C.
In 24 wells plate, slides were coated with PBS – Fibronectin (1/1,000), 2 h at 37 °C and wash twice with PBS.
Non adherent bovine cells (500,000 cells/well in 24 wells plate) were plated on Fibronectin coated slides 2 h at 37 °C, 5% CO₂ and then fixed in 500 μl PBS 3.7% Formaldehyde for 15 min at room temperature.
Slides were rinsed in PBS (300 μl - 3 washes) and permeabilized with PBS 0.2% Triton X-100 for 5 min.
Slides were washed in PBS (300 μl - 3 washes) and then blocked for 30 min at room temperature with 250 μl PBS 1% SVF and 1% BSA to prevent non-specific staining.
The slides were incubated with Mouse monoclonal anti-Ki67 (1:50) in 100 μl PBS 1% SVF and 1% BSA at room temperature for 40 min.
After washing in PBS 0.2% Tween (300 μl - 3 washes), the slides were incubated with Cy2 AffinyPure anti-mouse IgG (1:5,000) in 100 μl PBS 1% SVF and 1% BSA for 30 min at room temperature, in the dark.
Slides were subsequently washed in PBS 0.2% Tween (300 μl - 3 washes), mounted on slides and coverslippedwith ProLong Gold Antifade Reagent with DAPI.
Images of immunofluorescence staining were photographed with a camera attached to a fluorescent microscope and percentage of Ki67 positive cells was calculated.
This staining was repeated for three independent biological replicates (Figure 1).

Figure 1. Ki67 staining of bovine cells

Acknowledgments

This protocol was adapted form James et al. (2005). The Weitzman laboratory was supported by Fondation de France (FdF #2102), Association pour le Recherche contre le Cancer (ARC Fixe #4975, ARC-Equipement #7990) and Association for International Cancer Research (AICR, #08-0111).

References

James, D., Levine, A. J., Besser, D. and Hemmati-Brivanlou, A. (2005). TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development 132(6): 1273-1282.
Marsolier, J., Pineau, S., Medjkane, S., Perichon, M., Yin, Q., Flemington, E., Weitzman, M. D. and Weitzman, J. B. (2013). OncomiR addiction is generated by a miR-155 feedback loop in Theileria-transformed leukocytes. PLoS Pathog 9(4): e1003222.