# Comparison of preparation methods of rat kidney single-cell suspensions

## TL;DR

This paper introduces a new method for preparing high-quality single-cell suspensions from rat kidneys, which is important for various biological studies.

## Contribution

The novel contribution is the development of the stepwise enzymatic digestion (StE) method for rat kidney single-cell suspensions.

## Key findings

- The StE method yields higher harvest rates of glomerular cells and immune cells.
- The StE method results in higher cell viability and singlets rate compared to other methods.

## Abstract

Preparation of kidney tissue single-cell suspensions is the basis of single-cell sequencing, flow cytometry and primary cell culture, but it is difficult to prepare high quality whole kidney single-cell suspensions because of the complex structure of the kidney. We explored a technique called stepwise enzymatic digestion (StE) method for preparing a single-cell suspension of rat whole kidney tissue which contained three main steps. The first step is to cut the kidney into a homogenate. The second step is the digestion of renal tubules using Multi Tissue Dissociation Kit 2 and the last step is the digestion of glomeruli using type IV collagenase. We also compared it with two previous techniques, mechanical grinding method and simple enzymatic digestion method. The StE method had the advantages of high intrinsic glomerular cells and immune cells harvest rate, high singlets rate and high cell viability compared with the other two techniques. In conclusion, the StE method is feasible, highly efficient, and worthy of further research and development.