Blog

Using Akadeum’s Mouse RBC Depletion Microbubbles to enrich splenocytes for iNKT isolation

July 2021

Use of Akadeum’s gentle and effective Mouse RBC Depletion Microbubbles results in a highly enriched sample of splenocytes for iNKT isolation while maintaining the health and physiology of these precious few available cells.

Noemi Alejandra Saavedra-Avila, Ph.D., a Research Associate in the lab led by Steven Porcelli, M.D., at the Albert Einstein College of Medicine, used the Mouse RBC Depletion Kit from Akadeum Life Sciences in the hopes of finding a gentle and effective method that could replace the harshness of lysis reagents. With an interest in isolating a rare population of T cells, iNKT cells, which make up about 0.3% of their starting sample, it is critical to achieve maximal enrichment without damaging the precious few available cells. Akadeum’s microbubble approach to RBC depletion offers effective removal of contaminating RBCs using an exceptionally gentle process that maintains the health and physiology of delicate cells of interest.

Erythrocytes account for more than two thirds of all the cells obtained in Dr. Saavedra-Avila’s splenocyte preparations. This provides a tremendous opportunity for enriching other cell types through depletion of the unwanted RBCs. Using the Akadeum’s Mouse RBC Depletion Kit allowed Dr. Saavedra-Avila to do just that. The microbubbles eliminated RBC contamination as effectively as a traditional lysis buffer, with more than 95% of the RBCs cleared and a 97.1% pure population of splenocytes obtained (Figure 1). While the use of RBC lysis buffer cleared an equivalent percent of RBCs, it resulted in the presence of more debris (Figure 2A) and yielded lower viability in the remaining cells (Figure 2B).

Scope of the Project:

Objective:
Replace harsh lysis conditions with a gentler method for removing RBCs to enable enrichment of better quality iNKT cells

Kit Used:
Mouse RBC Depletion Kit from Akadeum Life Sciences

Procedure:
Spleens from humanized mice were obtained and manually dissociated. Following dissociation, the free splenocytes were passed through a 70 μm filter at which point erythrocytes were cleared using either an RBC lysis buffer or Akadeum’s Mouse RBC Depletion Kit. Following the elimination of RBCs, the samples were stained and analyzed via flow cytometry.

Key Takeaways:
Mouse RBC Depletion Microbubbles from Akadeum gently remove over 95% of RBCs while also generating significantly less debris and healthier cells.

Outcome:
Efficient removal of RBCs produced a 97.1% pure population of enriched splenocytes. Replacing the harshness of lysis buffer with the mildness of Akadeum’s Mouse RBC Depletion Kit resulted in cleaner samples with improved viability.

 

Figure 1: Akadeum’s Mouse RBC Depletion Microbubbles remove RBCs as effectively as treating samples with a lysis buffer.

 

Figure 2: Akadeum Mouse RBC Depletion Microbubbles yield more viable cells and significantly cleaner samples.

 

Akadeum’s Mouse RBC Depletion Kit delivers a highly enriched population of cells for downstream analysis using our protocol that is fast, easy, and gentle on cells. Ideal for processing delicate cells, Akadeum’s novel Buoyancy Activated Cell Separation (BACS™) occurs directly in the sample container without exposing cells to harsh chemicals.

If you are involved in cell isolation research, we’d love to hear from you! We are actively seeking new applications for our microbubble technology and would welcome the opportunity to learn more about your research and to discover if our microbubbles could help you in your work. Let’s schedule a time to talk!

Back to Top