AKLIDES® Nuk Tests

The AKLIDES® Nuk Human Lymphocyte Isolation (4278) kit is used for the isolation of human lymphocytes from whole blood.

The  AKLIDES® Nuk Human Lymphocyte Complete (4162) and the AKLIDES® Nuk Human Lymphocyte Complete Combi (4268) kits are used for the isolation of human lymphocytes from whole blood including the subsequent immunofluorescence staining of DNA repair foci. An established marker for the determination of DNA double strand breaks is γH2AX (4162), in addition the p53 binding protein 53BP1 can be used in combination with γH2AX (4268).

  • The kits described are specifically designed for standardized sample processing.
  • After venipuncture the samples should be processed within two hours.
  • γH2AX coated microparticles which generate fluorescence intensities within a certain range serve as reaction control
  • The evaluation can be done either visually or automatically using the AKLIDES® Cell Damage System.

A DNA double strand break in the cell triggers the kinase Ataxia Teleangiectasia Mutated which in turn phosphorylates the serine-139 residue of the histone subunit H2AX. The p53 binding protein 53BP1 binds to the phosphorylated H2AX (γH2AX) and regulates the repair of DNA double strand breaks. γH2AX and 53BP1 can be tagged by appropriate antibodies so that individual double strand breaks can be detected via secondary fluorochrome-labelled antibodies. The thus obtained fluorescent signal is visible as foci (pixel).

Duplicates are mandatory to ensure statistical reliability.  

Assay principle

Indirect immunofluorescence assay

Assay size

30 duplicates

Parameter

γH2AX (4162) / γH2AX and 53BP1 (4268)

Result

Quantitative determination of DNA repair foci

Incubation time

60 - 60 min

Sample material

At least 3 ml of whole blood

For significant determination of DNA repair foci the quality of the samples is crucial. This can be assured by standardized processing with the materials given. Furthermore, slides with predefined wells enclosed within the kit provide simple assignment of samples.

Heydenreich, J., Otto, C., Mayer, F. & Carlsohn, A.  Reliability of a Fully Automated Interpretation of γH2AX Foci in Lymphocytes of Moderately Trained Subjects under Resting Conditions. Journal of Nutrition and Metabolism 2014 Jul 24; Article ID 4783241-7.

 

Reddig, A., Lorenz, S., Hiemann, R., Guttek, K., Hartig, R., Heiserich, L., Eberle, C., Peters, V., Schierack, P., Sack, U., Roggenbuck, D. & Reinhold, D. Assessment of Modulated Cytostatic Drug Resistance by Automated γH2AX Analysis. Cytrometry 2015; Part A, 87A 724-732.

 

Runge, R., Hiemann, R., Wendisch M., Kasten-Pisula U., Storch K., Zoephel K., Fritz C., Roggenbuck D., Wunderlich G., Conrad K. & Kotzerke J. Fully automated interpretation of ionizing radiation-induced γH2AX foci by the novel pattern recognition system AKLIDES®. International Journal of Radiation Biology 2012 May; 88(5): 439-47.

 

Willitzki, A., Lorenz, S., Hiemann, R., Guttek, K., Goihl, A., Hartig, R., Conrad, K., Feist, E., Sack, U., Schierack, P., Heiserich, L., Eberle, C., Peters, V., Roggenbuck, D. & Reinhold, D. Fully automated analysis of chemically induced γH2AX foci in human peripheral blood mononuclear cells by indirect immunofluorescence. Cytometry 2013; Part A, 5-9-2013; Volume 83, Issue 11, Page 1017-1026.

 

Willitzki, A., Hiemann, R., Peters V., Sack U., Schierack P., Rödiger S., Anderer U., Conrad K., Bogdanos D. P., Reinhold D. & Roggenbuck D. New platform technology for comprehensive serological diagnostics of autoimmune diseases. Clinical and Developmental Immunology 2012; Article ID 284740.