Cost Effective And Robust Method Of Staining Intravascular Leukocytes In Vivo With CD45 Antibody

September 20, 2016

Pediatrics
UW-Madison
Postdoc

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Company:

BioLegend

Product Name:

anti-mouse CD45-PE (clone 30-F11)

Catalog Number:

103106

When interrogating populations of leukocytes in an organ or tissue it is often important to distinguish exudate or resident populations from populations present in capillaries. The capillary beds of lungs for instance have a high concentration of neutrophils that remain in the organ unless the mouse is bled and the heart transfused thoroughly with PBS. Using the method described by Anderson et al. 2014 (Nature Protocols), I have used anti-CD45-PE (clone 30-F11) from BioLegend with great success to stain capillary leukocytes while leaving leukocytes in parenchyma and airways unstained. The PE fluorescence was maintained through lung dissociation and enzymatic digest. This particular reagent from BioLegend is especially suited for this technique because it costs much less than other fluorescent conjugated antibodies; this technique uses micrograms of antibody per each mouse. The technique of staining intravenous leukocytes in vivo has been well validated for lung tissue, but may require antibody titration or other optimization for other tissue.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Lung samples from (in this case naive) mice

Primary Incubation

In vivo (see reference); 2ug of anti-CD45-PE injected intravenously (at 4 ug/ml) before euthanizing mice

Blocking Agent

None

Secondary Incubation

After tissue processing and cell preparation, stain for surface markers as normal

Tertiary Incubation

None

Detection

Fluoresecence in flow cytometry (could be used for fluorescent microscopy)

Results Summary

The data shown is representative of naive mice where all cells in peripheral blood (only neutrophils and monocytes are highlighted) were stained with the anti-CD45 that was injected intravenously 3-5 minutes before euthanizing mice (the peripheral blood sample is an experimental positive control). For the lung tissue three populations are shown: alveolar macrophages (a resident population in the lungs), neutrophils and inflammatory monocytes (both only present in lung capillaries under non-inflammatory circumstances). Because anti-CD45 doesn't block the epitope of anti-CD45.2, anti-CD45.2 was used to stain ex vivo to validate intravenous staining. The expression of CD45 various among leukocytes, so an angled gate was necessary to gate cells positively and negatively staining with the intravenously administered antibody.

Additional Notes

Later replicates of this experiment with infected lungs showed greater resolution when lungs where rinsed immediately in a large volume of PBS and processed in double the typical volume of digest buffer because excess antibody tends to cross-over and stain cells not in capillaries. (This is more noticeable for DCs and macrophages). Can inject antibody into tail or retro-orbital veins.

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Summary

The Good

Reagent is very inexpensive for a fluorescent antibody, which is important because 2 ug (10 ul of the stock) is given per mouse. PE is very stable through tissue processing. Can stain later with either CD45.1 or CD45.2 ex vivo after i.v. stain in vivo.

The Bad

PE may not be the best fluor for this marker in certain experimental designs.

The Bottom Line

The technique for injecting antibody intravenously is useful for studying in vivo populations, but can get very expensive because micrograms of antibody are needed for each mouse. This reagent currently costs $0.35 per microgram (when purchasing 200 ug) making it probably the most cost effective reagent for this technique.

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