Pre-Enrichment of Innate Lymphoid Cells Prior to Sorting Improves Efficiency
- Document # 27137
- Version 1.0.0
- Sep 2018
Isolating rare cell types by flow sorting alone can be time consuming, especially when working with large sample volumes or rare cell types. Starting your sort with pre-enriched populations allows you to obtain your rare cells faster than by using fluorescence-activated cell sorting (FACS) alone, saving you time and money.
Pre-enrichment of your target population can be quickly achieved by depleting unwanted cells. For example, you can use immunomagnetic cell separation technologies to pre-enrich cells that are then ready to be labeled with antibodies for immediate cell sorting by flow cytometry.
Here, the isolation of innate lymphoid cells (ILCs) is used as an example to show how pre-enrichment prior to FACS can significantly reduce sorting times and increase final cell purities when working with rare and complex cell types.
Innate lymphoid cells (ILCs) are important effector cells in innate immunity and have essential functions in immune regulation, inflammation, and tissue homeostasis. In healthy individuals, ILCs are extremely rare, comprising <0.1% of cd45+ leukocytes in mouse and human peripheral blood.1,2 The low frequency and lack of unique surface markers for ILCs create challenges when it comes to their identification, isolation, and characterization. ILCs are typically isolated using multicolor FACS; however, this method is time-consuming, expensive, and can result in low cell recovery. We describe a method to drastically reduce the time required to isolate all ILC groups (ILC1, ILC2 and ILC3).
Single-cell suspensions were prepared by washing unprocessed leukapheresis samples twice with PBS containing 2% fetal bovine serum (FBS). Following staining of the single-cell suspensions with fluorescently labeled antibodies, ILCs were isolated by FACS. Alternatively, single-cell suspensions were pre-enriched for target cells using the EasySep™ Human Pan-ILC Enrichment Kit (Catalog #17975) prior to staining and FACS. In brief, ILCs were enriched by immunomagnetic negative selection, where unwanted cells were targeted for removal by antibody complexes and magnetic particles. Final purities, recoveries, and protocol times were compared between the two isolation methods.
To evaluate the effect of pre-enrichment with EasySep™ on ILC isolation, unenriched and EasySep™-enriched samples were stained with fluorescently labeled antibodies, and ILCs were identified as CD45+, lineage-negative (CD1a, CD3, CD11c, CD14, CD19, CD34, CD123, TCRαβ,TCRγδ, BDCA-2, FcεR1α, CD94, CD4, CD16), and CD127+ by flow cytometry. ILC1, ILC2 and ILC3 populations were further defined based on CRTH2 and CD117 expression. As shown in Figure 1, the EasySep™ kit significantly increased the purity of ILCs prior to FACS.
Figure 1. Pre-Enrichment with EasySep™ Improves ILC Starting Purity Prior to FACS
(A) Starting with a fresh leukopheresis sample, unenriched and EasySep™- enriched cells were stained and gated on WBC, viable cells, CD45+ , Lin− and CD127+ . ILC1, ILC2, and ILC3 subsets were identified based on expression of CRTH2 and CD117. Representative FACS plots are shown. (B) The ILC frequency of the unenriched and EasySep™-enriched sample was 0.07% and 53.6% of CD45+ cells, respectively (Unenriched n = 20, EasySep™-Enriched n = 22; data represents the median ± SEM).
To evaluate the efficiency of ILC sorting from unenriched and EasySep™-enriched leukapheresis samples, FACS time and ILC purities were compared (Figure 2).
Figure 2. Pre-Enrichment with EasySep™ Significantly Reduces the Time Required to Obtain Pure ILCs
Starting with a fresh leukapheresis sample, ILCs were isolated in parallel from an unenriched or an EasySep™-enriched sample. (A) In an unenriched sample, ILC frequency was assessed by flow cytometry at the start and after one round of FACS. (B) In an EasySep™-enriched sample, ILC frequency was assessed immediately after EasySep™-enrichment, and again after one round of FACS. (C) Corresponding purities and FACS times at each stage are reported.
Starting with 7.5 x 107 total cells and a pre-FACS purity of 0.1%, isolation by FACS alone resulted in a final ILC purity of 74% and took approximately 120 minutes of sort time. A second round of sorting was required in order to obtain a final purity of 97% (Data not shown). Extrapolating from these results, it would take approximately 3,200 minutes of sort time to process 2 x 109 cells. Conversely, starting with 2 x 109 cells from the same donor, pre-enrichment by EasySep™ increased the pre-FACS ILC frequency from 0.1% to 27%. Flow sorting time for this EasySep™-enriched sample was reduced to 12 minutes, with a final Lin− CD127+ purity of 99%. As shown in Figure 3, sorted ILC1, ILC2 and ILC3 subsets from EasySep™-enriched samples remain functional as assessed by cytokine secretion.
Figure 3. EasySep™-Enriched ILCs are Functional
ILC1, ILC2 and ILC3s sorted from EasySep™-enriched samples were cultured independently in a serum-free expansion medium, with or without stimuli. After 6 days, supernatants from ILC1 (IL-12 and IL-15), ILC2 (IL-2 and IL-33) and ILC3 (IL-2, IL-1β and IL-23) cultures were collected and analyzed for the secretion of IFNγ, IL-13, and IL-17A, respectively, by ELISA.
By using EasySep™ to pre-enrich for ILCs, it is possible to process large sample sizes to isolate distinct ILC subsets. The short protocol times of EasySep™ and RosetteSep™ kits provide an easy means of streamlining the cell isolation step in a given assay. In this case study, we demonstrate how a negative selection protocol can quickly deplete unwanted cells to enrich for rare untouched ILCs that are ready for analysis or cell sorting. By combining FACS with EasySep™, the overall cell isolation process resulted in greater purity in a significantly shorter period of time.
- Mjösberg JM et al. (2011) Human IL-25- and IL-33-responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161. Nat Immunol 12(11): 1055-62.
- Halim TYF & Takei F. (2014) Isolation and characterization of mouse innate lymphoid cells. Curr Protoc Immunol 106(5): 3.25.1-13.