Excellent Antibody for Detecting Ki67 in Mouse

Charite Strasse 1
Deutsches Rheuma-Forschungszentrum
Research Scientist

Overall

Quality of Results

Ease-of-Optimization

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Company:

Miltenyi Biotec

Product Name:

Ki-67 Antibody, anti-human/mouse, REAfinity™

Catalog Number:

130-117-691

Clone Number:

REA183

In different healthy and pathological contexts CD4 T cells differentiate into a range of specialized interleukin-secreting subsets. In mouse models of aging and autoimmune diseases we study the plasticity and effector functions of CD4 T helper cells. We used FITC anti-ki67 antibody to detect proliferation in different cytokine-producing CD44+CD4 T cell subsets from old C57Bl/6J mice.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Splenocytes of wild-type 12 months-old C57Bl/6J mouse, incubated in complete culture medium at 37 °C with PMA/ionomycin for 4h and brefeldin A for the last 3 hours.

Primary Incubation

Surface staining with anti-CD4 Al647 and CD44 BV785 in PBS 0.5% BSA incubated 30min at 4°C. Washed twice in PBS and incubated with L/D fixable aqua dye 20 min at 4°C, then washed twice in PBS.

Blocking Agent

Clone 2.4G2 (Fc block)

Secondary Incubation

Cells are then fixed and permeabilized with commercial kit to detect transcription factors (30 min at 4°C with fixative, and 15 min at 4°C with permeabilization buffer). Cells were incubated with FITC anti-Ki67 at 1/200 and different anti-cytokine antibodies in permeabilization buffer for 30 min at 4°C and washed once with permeabilization buffer and once with PBS BSA 0.5%.

Tertiary Incubation

N/A

Detection

Flow cytometry, blue laser

Results Summary

I could quantify Ki67 positive cells in relation with cytokine expression in the spleen.

Image Gallery

Summary

The Good

The antibody works well, it is an excellent option to use in mouse compare to other anti-Ki67 antibodies rise in mouse and possibly interfering with anti-IgG staining to detect antibody producing cells.

The Bad

None

The Bottom Line

Excellent option

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