isolation and preparation of mice splenic single-cell suspension for flow cytometry analysis

<meta charset="utf8"></head><body>使用流式细胞术从初始 CD4+ T 细胞中分化 Th17 细胞

After leaving the thymus, naive CD4+ T lymphocytes pass through secondary lymphoid organs. Antigen-presenting cells that transmit homologous antigens to na&#239;ve CD4+ T cells activate them, starting a series of differentiation programs that eventually result in the production of highly specialized T helper (Th) cell lineages1. Interleukin 17 (IL-17) production characterizes Th17 cells, a subpopulation of pro-inflammatory Th cells2. Th17 cells play a role in the host&#39;s defense against extracellular pathogens and in the pathogenesis of many autoimmune diseases, such as autoimmune uveitis and multiple sclerosis. Signals from T-cell receptors and cytokines IL-6 and transforming growth factor-&#946; (TGF-&#946;) induce the differentiation of naive T cells into Th17 cells through the phosphorylation of signal transducer and activator of transcription (STAT)33. STAT3 is further amplified in a positive feedback loop through signaling mediated by IL-23 and IL-214,5. Phosphorylation of STAT3 can induce the expression of the transcription factors ROR&#947;t and ROR&#945;, which act as master switches regulating the cytokine profile of IL-17A, IL-17F, IL-21, and IL-22 in Th17 cells6. However, it has been reported that IL-6- and TGF-&#946;-induced Th17 cells are insufficient to trigger autoimmune diseases, which require co-stimulation by IL-23 or separate co-stimulation of IL-6, IL-1&#946;, and IL-23 in the absence of TGF-&#946;7,8.
Th17 subsets that cannot effectively induce experimental autoimmune encephalomyelitis (EAE) are sometimes referred to as non-pathogenic Th17 while Th17 subsets that can induce EAE are known as pathogenic Th179. Current studies have shown that although pathogenic Th17 and non-pathogenic Th17 co-express the core transcription factor ROR&#947;t, there are great differences in the ability to produce IL-17A and pro-inflammatory and anti-inflammatory properties10. In addition to the high expression of ROR&#947;t, CCR6, STAT4, and RUNX4, which are common characteristic transcription factors of Th17, pathogenic Th17 cells also show additional gene signal expression characteristics related to the disease, such as TBX21, IFN-&#947;, and CXCR3, which have the characteristics of Th1 cell subsets. Pathogenic Th17 cells can secrete high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-&#947;, TNF-&#945;, and other cytokines11,12. The phenotype of non-pathogenic Th17 cells is unstable, and only under the stimulation of CD3 and cytokine IL-2 can some of these cells differentiate into pathogenic Th17 cells. Therefore, in common clinical disease models such as rheumatoid arthritis, multiple sclerosis, and acute respiratory distress syndrome, pathogenic Th17 cells primarily exert pathogenic effects.
Pathogenic and non-pathogenic Th17 cells can differentiate in vitro under the influence of different cytokines. In recent years, several studies have proposed methods for inducing the differentiation of Th17 cells using different types and concentrations of cytokines. Th17 cells are stimulated by a combination of IL-6, IL-1&#946;, and IL-2313,14,15,16. It has been proved that IL-6 and TGF-&#946;, two cytokines necessary for Th17 cell differentiation, synergistically regulate the expression of ROR&#947;t and Th17 cell differentiation by interacting with two different conserved non-coding DNA sequences at the Rorc gene locus17. The stable phase of pathogenic Th17 cells is mainly maintained by IL-2318,19. IL-23 binds to its receptor and activates the JAK-STAT signaling pathway20, thereby causing phosphorylation of Jak2 and Tyk2 and promoting phosphorylation of STAT1, STAT3, STAT4, and STAT5. IL-4 and IFN-&#947; are negative regulators of this pathway. However, studies have shown that IL-1&#946; may positively regulate the transcription of Ror&#945; and Ror&#947;t through the mTOR pathway to maintain the stability of the Th17 cell phenotype21.
Due to the heterogeneity of numerous studies, we chose the induction protocols for pathogenic and non-pathogenic Th17 cells from the latest research as controls22. The results indicate that, assuming that everything is carried out according to this protocol, after 5 days of culture under the condition of generating pathogenic Th17, more than 90% of the surviving cells can be pathogenic Th17 cells.

<meta charset="utf8"></head><body>作者聚焦：使用细胞因子浓度调节实现 Th17 细胞的高纯度体外分化

小鼠幼稚 CD4+ T 细胞体外分化为致病性 Th17 细胞

Our research objective is to target the directed differentiation of Th17 cells, and we aim to explore a simple method for re-intro differentiation of Th17 cells, which serves as a traditional condition for re-intro experiments related to some disease associated Th17 cells. Many studies have mentioned the methods for reintroduction of Th17 cells, and the recent research has focused on small molecular inhibitors and other stimuli. However, the purity of in-utero induction of almost all studies has not been stable above 50%Many new techniques have been applied to the screen and circulation of Th17 cells, such as flow cytometry sorting of the Th17 cells and the magnetic bead sorting for Th17 cells.The key point is that we found a simple method to induce Th17 cell differentiation, by adding different concentrations of to the medium, and its differentiation purity can be close to 90%Our laboratory, we are studying the changes in the imino-microenvironment in self-stasis models, including the change in immune cells and the direction of immune imbalance, and so on.

用于流式细胞术分析的小鼠脾单细胞悬液的分离和制备

isolation-preparation-mice-splenic-single-cell-suspension-for-flow

Research

JoVE Journal

Immunology and Infection

Isolation and Preparation of Mice Splenic Single-Cell Suspension for Flow Cytometry Analysis

In Vitro Differentiation of Naive CD4+ T Cells into Pathogenic Th17 Cells in Mouse

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have been linked to a wide range of diseases in recent times, including multiple sclerosis (MS), rheumatoid arthritis, acute respiratory distress syndrome (ARDS), sepsis, and other autoimmune disorders. However, the currently known in vitro differentiation protocols have difficulty achieving high purity of pathogenic Th17 cells, with the induction efficiency often below 50%, which is a key challenge in in vitro experiments. In this protocol, we propose an enhanced in vitro culture and differentiation protocol for pathogenic Th17 cells, which is used to directly differentiate naive CD4+ T cells isolated from mouse spleens into pathogenic Th17 cells. This protocol provides detailed instructions on splenocyte isolation, purification of naive CD4+ T cells, and differentiation of pathogenic Th17 cells. Through this protocol, we can achieve a differentiation purity of approximately 90% for pathogenic Th17 cells, which meets the basic needs of many cellular experiments.

This protocol describes the differentiation of na&#239;ve CD4+ T cells into pathogenic Th17 cells in vitro. Specifically, when combined with a multi-parameter flow cytometry-based approach, 90% purity of pathogenic Th17 cells can be obtained from na&#239;ve CD4+ T cells using this differentiation method.

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have ...

科研

免疫与感染

To begin, place the euthanized mouse in a supine position on the clean bench. Cut along the midline of the mouse abdomen and separate the abdominal structures layer by layer. Using tweezers, gently open the greater omentum and stomach.Then gently pull out the spleen with the gastrosplenic ligament and bluntly separate it from surrounding tissues and ligaments to obtain an intact spleen. Place a 70 micron cell filter in a 100 millimeter sterile culture dish. Add the spleen to the cell strainer and crush it with a syringe plunger.Add five to eight milliliters of homogenate flushing fluid to push the splenic cells through the filter into the culture dish. Centrifuge the filtrate at 450g for five minutes at room temperature and discard the supernatant. Re-suspend the pellet with the sample diluent provided with the lymphocyte separation kit.After counting the cells on a hemocytometer, adjust the cell concentration to 2 X 10 to the power of eight cells per milliliter. In a centrifuge tube, take the same amount of lymphocyte separation solution as the tissue single cell suspension. Carefully pipette the single cell suspension onto the surface of the lymphocyte separation solution and centrifuge at 800g for 30 minutes at 25 degrees Celsius.After centrifugation, observe the four layers in the tube from top to bottom. Using a pipette, carefully draw the annular milky white lymphocyte layer into another tube. Add 10 milliliters of cleaning solution to the tube to mix the cells.Centrifuge the cell suspension at 250g for five minutes. Discard the supernatant and re-suspend the pellet in 10 milliliters of RPMI 1640 medium for cell counting.

Purification and Analysis of Mice Naive CD4+ T-Cells Using Magnetic Bead-Based Negative Selection

To begin, prepare a mouse splenic single cell suspension with one times 10 to the power of eight cells per milliliter in a volume of 0.1 to two milliliters. Add 50 microliters of rat serum to the sample. Transfer the sample to a five milliliter polystyrene round bottom tube.Add 50 microliters of isolation cocktail to the sample. Mix well and incubate for 7.5 minutes at room temperature. Now add 50 microliters per milliliter of depletion cocktail.Mix well and incubate for 2.5 minutes. Meanwhile, vortex the magnetic beads to ensure even dispersion. Add 75 microliters of the magnetic beads to the sample.Mix well and incubate for 2.5 minutes. Top up the sample with RPMI 1640 medium to a final volume of 2.5 milliliters and mix several times. Then place the tube into the magnet for 2.5 minutes.Pick up the magnet and invert it with the tube in one continuous motion, pouring the enriched cell suspension into a new tube. Determine the cell number using a hemocytometer. Perform flow cytometric analysis for naive CD four positive T-cell before and after isolation.After gating, transfer the cell suspension to a new centrifuge tube. Centrifuge at 400G for five minutes and discard the supernatant. Resuspend the pellet in 200 to 500 microliters of RPMI 1640 medium supplemented with 10%FBS.For each one milliliter of cell culture, add two microliters of leukocyte activation cocktail and mix. Culture the cells in a carbon dioxide incubator at 37 degrees Celsius with saturated humidity for four to six hours.

使用基于磁珠的阴性选择纯化和分析小鼠幼稚 CD4 + T 细胞

In Vitro Induction of Pathogenic Th17 Cells into Naive CD4+ T-Cells of Mice

To begin, remove the PBS from the pre-coated 24-well plate. Resuspend the enriched mouse splenic naive CD4+T cells in Th17 cell culture medium and dispense into different wells of a 24-well plate pre-coated with anti-CD3. Add Th0 cell culture medium, classical non-pathogenic Th17 culture medium, and classical pathogenic Th17 culture medium for contrast into the remaining wells of the plate.Add one microliter of anti-CD28 solution to each well. Culture the cells in a 5%carbon dioxide incubator at 37 degrees Celsius for five days. On day two, replace half of the cell culture supernatant medium with fresh culture medium.Observe the cells under an optical microscope on day five. Collect the cell supernatants from each group and cryopreserve them at 80 degrees Celsius. Naive CD4+T cells were cultured with Th0 medium, and Th17 differentiation medium for five days, resulting in T cells showing cluster growth in both media.Flow cytometry analysis revealed that 90%of naive CD4+T cells successfully differentiated into pathogenic Th17 cells under the stimulation of a new Th17 cell culture medium.