CD45 is a type I transmembrane molecule found on the surface of all nucleated hematopoietic cells, and is an ideal target for mouse immune cell (i.e. leukocyte) isolation due to its high level of expression on these cells across different mouse strains.^{1, 2} Immune cells found in specific tissue microenvironments, also referred to as tissue-resident immune cells (TRICs), are an important cell source for basic research, drug screening, preclinical animal studies, tumor models, etc. However, isolating TRICs can be challenging due to the presence of non-immune, tissue-derived cells and cellular debris following tissue dissociation. This results in reduced leukocyte starting frequency, a common obstacle that can slow down the isolation process and limit the identification of small but critical leukocyte subsets.

Isolating cells based on their CD45⁺ expression is also a popular approach to obtain cells that infiltrate tumors and constitute the tumor microenvironment (TME). These tumor-infiltrating leukocytes (TILs) are important for researchers working with tumor models, or performing downstream applications such as drug screening, cell culture, or single cell analysis. However, isolating and analyzing TILs can be difficult due to their relatively low frequency combined with the heterogeneity and abundance of tissue debris in tumor samples.

To overcome the challenges associated with CD45⁺ immune cell isolation, we have developed two mouse CD45⁺ isolation kits that enrich for CD45⁺ immune cells in as little as 18 minutes using EasySep™ technology—a simple and rapid immunomagnetic selection method that doesn’t require columns. Additionally, EasySep™ can either enrich for, or deplete CD45⁺ leukocytes from a variety of mouse tissues while preserving cell function. This tech tip summarizes key considerations to help you select the most appropriate EasySep™ CD45⁺ isolation kit for your particular starting tissue.

Which EasySep™ Kit Should I Use to Obtain CD45+ Cells from Mouse Tissue?

We currently offer two EasySep™ positive selection kits that target the mouse CD45 antigen:

1. EasySep™ Mouse CD45 Positive Selection Kit (Catalog #18945)
2. EasySep™ Mouse TIL (CD45) Positive Selection Kit (Catalog #100-0350)

Some key considerations for choosing the optimal EasySep™ kit to isolate CD45⁺ mouse leukocytes are listed below. Please refer to Table 1 for a complete list of differences between the two kits.

• Sample type: healthy vs. tumor tissue
• Starting frequency of CD45⁺ cells: high (> 30%) vs. low (< 30%)
• Target cells: depletion vs. isolation/obtention of CD45⁺ cells

When Should I Use EasySep™ Mouse TIL (CD45) Positive Selection Kit?

The EasySep™ Mouse TIL (CD45) Positive Selection Kit is recommended to isolate leukocytes from solid mouse tumors or tissue samples where the starting frequency of CD45⁺ cells is low (< 30%), as it should yield better results and require minimal to no optimization. In some cases, optimization may be required due to the heterogeneity of mouse tumors and the specific downstream applications of each user. In order to provide flexibility to the user, this kit provides protocol recommendations (at Step 1 of “Selection Protocol” in the Product Information Sheet) to skew the isolation toward high purity or high recovery. Please refer to Pages 3–4 of the Product Information Sheet for further details.

When Should I Use EasySep™ Mouse CD45 Positive Selection Kit?

The EasySep™ Mouse CD45 Positive Selection Kit is recommended for the isolation of leukocytes from healthy tissues such as mouse spleen and lungs, or tissues where the start frequency of CD45⁺ cells is high (> 30%). Use of this kit with other tissues may require optimization. Please contact techsupport@stemcell.com for more information.

When using the EasySep™ Mouse CD45 Positive Selection Kit to process debris-rich tissues with low (< 30%) CD45⁺ start frequency, the following methods of optimization can be applied:

• Perform tissue cleanup using an appropriate density gradient medium
• Titrate the Selection Cocktail down
• Increase the number of magnetic separations
• Contact techsupport@stemcell.com for further assistance

For customers who have already optimized CD45⁺ cell isolation from tumor samples with the EasySep™ Mouse CD45 Positive Selection Kit, it is recommended to continue using this kit. For new customers, the EasySep™ Mouse TIL (CD45) Positive Selection Kit should yield better results and require less/no optimization. See the table below for a summary of the differences between the two kits.

Table 1. EasySep™ Kit Comparison to Isolate CD45⁺ Leukocytes

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