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Ki67 Immunofluorescence on Bovine Cell Lines   

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Original research article

A brief version of this protocol appeared in:
PLOS Pathogens
Apr 2013

Abstract

This is a rapid protocol to test the effects of drugs treatment on bovine cell replication using Ki67 staining. Ki67 is associated with cell proliferation and is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent from resting cells (G0).

Keywords: Ki67, Immunofluorescence, Bovine lymphocytes

Materials and Reagents

  1. 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)
  2. RPMI 1640 (Gibco)
  3. Fetal calf serum (heat-inactivated)
  4. Penicillin/streptomycin
  5. Fibronectin (Sigma-Aldrich, catalog number: F1141 )
  6. Formaldehyde (Sigma-Aldrich, catalog number: F8775 )
  7. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  8. SVF (PAA Laboratories GmbH, catalog number: A15-101 )
  9. BSA (Sigma-Aldrich, catalog number: A2153 )
  10. Mouse monoclonal anti-Ki67 (1:50) (Abcam, catalog number: ab10913-1 )
  11. Cy2 AffinyPure anti-mouse IgG (1:5,000) (Jackson ImmunoResearch Laboratories, catalog number: 715-225-150 )
  12. Tween 20 (Biosolve, catalog number: 2045 2335 )
  13. ProLong Gold Antifade Reagent with Dapi (Life Technologies, Invitrogen™, catalog number: P-36931 )
  14. DAPI

Equipment

  1. Slides (Knittel Glass, catalog number: KN00010025787 )
  2. 37 °C, 5% CO2 cell culture incubator
  3. 24 wells plate
  4. Fluorescent microscope (Leica Microsystems, model: Inverted 6000 )

Procedure

  1. 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.
  2. In 24 wells plate, slides were coated with PBS – Fibronectin (1/1,000), 2 h at 37 °C and wash twice with PBS.
  3. 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.
  4. Slides were rinsed in PBS (300 μl - 3 washes) and permeabilized with PBS 0.2% Triton X-100 for 5 min.
  5. 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.
  6. 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.
  7. 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.
  8. Slides were subsequently washed in PBS 0.2% Tween (300 μl - 3 washes), mounted on slides and coverslippedwith ProLong Gold Antifade Reagent with DAPI.
  9. Images of immunofluorescence staining were photographed with a camera attached to a fluorescent microscope and percentage of Ki67 positive cells was calculated.
  10. 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

  1. 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.
  2. 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.   
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Marsolier, J., Medjkane, S., Perichon, M. and Weitzman, J. B. (2013). Ki67 Immunofluorescence on Bovine Cell Lines. Bio-protocol 3(21): e958. DOI: 10.21769/BioProtoc.958.
  2. 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.
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