Utilization of CD45.1 as a Marker of Donor Leukocytes in Recipient CD45.2 Mice in a Bone-Marrow Transfer Chimeric Experiment

November 24, 2015

Surgery
George Washington University, Washington DC
Post-doctoral Fellow

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Company:

Affymetrix/eBioscience

Product Name:

Anti-Mouse CD45.1 FITC (Ly5.1)

Catalog Number:

11-0453-85

Info: View Product View Product Specs

Browse Similar Products: CD45.1 Antibodies

CD45 is common antigen expressed in all leukocytes. It has two different alleles, CD45.1 and CD45.2, which are functionally identical. We transferred bone marrow from CD45.1 mice (donor) to recipient CD45.2 mice in two different treatment groups. After 4 weeks of bone marrow transfer, recipient mice from both groups were sacrificed and splenocytes were isolated and stained with anti-CD45.1 FITC and anti-CD45.2 PE conjugated antibody to analyze the percentage of host vs. donor cells in both groups.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Mouse splenocytes, 4 weeks after bone marrow transfer

Primary Incubation

CD45.1 (Ly5.1) mice were sacrificed and ~10e7 bone marrow cells were injected through the tail vein into recipient CD45.2 mice. After 4 weeks the recipient mice were sacrificed and splenocytes isolated. The splenocytes were stained with antiCD45.1 FITC and anti CD45.2 PE conjugated antibody (1:50) along with anti- CD8APC and anti-CD4 Percpcy5.5 antibody (1:100 conc.) Cells were kept in a cocktail containing the antibodies in mentioned conc. for 30 mins at 4 degree C. Then the cells were washed twice with PBS and resuspended in 500uL of FACS buffer. Cells were analyzed in flow cytometer for the percentage of CD45.1 and CD45.2 positive cells.

Blocking Agent

NA

Secondary Incubation

NA

Tertiary Incubation

NA

Detection

Flow cytometry

Results Summary

Leukocytes were either CD45.1 positive or CD45.2 when gated on either CD4+ or CD8+ T cells. The percentage of CD45.1, (donor cells) though less than CD45.2, was significant enough to depict a successful bone marrow transfer and chimera generation.

Additional Notes

All leukocytes should be either CD45.1 or CD45.2 positive if the bone marrow transfer is succesfully done.

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Summary

The Good

Chimera experiments are of immense importance for knock-out models and CD45.1/CD45.2 is a great model for such purposes. Staining is the relatively easier part of the experiment and a reliable technique to quantify transferred cells.

The Bad

The entire chimera experiment is difficult and time consuming but the staining for the anti CD45.1/CD45.2 antibody is simple

The Bottom Line

Good, accurate staining achievable with anti-CD45.1 FITC and CD45.2 PE conjugated antibody to differentiate Ly5.1 and Ly5.2 mouse leukocytes.

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