Referee 1's Review:

In this manuscript Ieronimakis and colleagues report on the isolation and culture of mouse skeletal muscle endothelial cells. To isolate these cells they use Sca+, CD31+ CD34(dim) and CD45- as criteria for FACS sorting. The cells are cultured and characterized as functional endothelial cells by the presence of several markers, their ability to form vascular cords/tubes and the ability to produce NO (DAF-2A, eNOS verified by antibody). The description of the method is extensive and clear

1. The authors use stem cell antigen-1 (Sca-1) together with CD31 and CD34(dim) and absence of CD45 as criteria for the isolation of muscle endothelial cells. The study does not make clear how many percent of muscle endothelial cells in vivo are positive of Sca-1 and whether these cells represent a specific subtype or vessel subtype. The introduction does not provide references to substantiate this. It makes it difficult to discriminate between vascular progenitor cells that are differentiating in endothelial cells and the quiescent endothelial cells that make up the stable vasculature of the muscle. The authors should further elucidate this aspect.

2. While the description is clear and may be helpful to isolate and culture cells derived from striated muscle that have endothelial characteristics, there has been made no attempt to verify whether these endothelial cells have indeed muscle specific characteristics nor whether such characteristics are maintained during culturing. How do we know that they are different from other continuous endothelia, e.g. from skin or connective tissue?

3. It remains unclear whether the EC isolated and cultured are EC from arterioles, capillaries, postcapillary venules, small arteries or veins. Have lymphatic EC been excluded within the population? Furthermore, does a selection of cells occur during subculturing? Have the authors any estimation how many cells detach and are lost?

4. The isolation of muscle endothelial cells may be of interest for studying the metabolic syndrome and insulin resistance. However, how certain are the authors that the cells obtained do not have metabolic syndrome themselves? The ambient oxygen generates radicals, the cells are cultured in high D-glucose, and the L-carnitine concentration is only 10% of what endothelial cells normally experience, which causes a reduction of B-oxidation of free fatty acids.

Although this reviewer does not expect that all items of 2,3 and 4 are fully elucidated, one would be appropriate to give the paper some added value, and the other aspects should be appropriately discussed.

5. The paper is rather widely written and could -without loss of information- be condensed.

Minor point.
Isolation procedure [As the pages have not been numbered I have counting them from the beginning the abstract page being page 2.]
Page 6 under cell isolation: The technical description is in general clear. Two aspects can be further clarified.
"Muscle was minced": how (e.g. with scissors) and how small were the pieces.
"passed through 70 um followed by 40 um nylon filters" Please indicate how much volume over how many mm2 were used, and whether the filters were washed to increase the recovery.
[page 7, under culture: Please confirm that the standard polystyrene tissue cultures dishes were not coated.]

Suitability for protocols.
The manuscript is well suitable for making a protocol.

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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.