分离及小鼠淋巴细胞的激活
Summary
Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.
Abstract
B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.
Introduction
Mature lymphocytes generally exist in the resting state if there is no pre-existing infection or inflammation in the individual. Therefore, it is important to retain the naïve status of lymphocytes during the isolation process before performing in vitro or in vivo functional assays. The key to ensuring consistent and reproducible results is to limit any unnecessary manipulation of the cells.
Magnetic cell sorting utilizes antibodies and microbeads to label cells so as to enrich the cell population of interest. With this approach, there are two purification strategies: positive enrichment and negative depletion. Positive enrichment enriches the cell population of interest using an antibody that binds to the target cells. Negative depletion, on the other hand, depletes non-target cells, leaving the cell population of interest. In our lab, we prefer negative depletion to positive enrichment because the binding of antibodies to the target cells could potentially alter cell features and behavior. In fact, many established cell surface markers suitable for the isolation of a particular cell population are also functional receptors.
Magnetic cell sorting not only yields highly pure populations of viable target cells, it is also less time-consuming and avoids the cellular stress induced by high-pressure flow used in fluorescence-activated cell sorting (FACS). By labeling the unwanted cell populations and depleting them using a magnetic separation column, we are able to perform rapid cell isolation without compromising the viability of the target cell population. In this protocol, we demonstrate the use of negative depletion strategies to purify naïve B cells or T cells.
Protocol
Representative Results
Discussion
在这个协议中，我们展示了从淋巴器官淋巴细胞净化的过程。使用磁珠分选的细胞纯化是产生可行的，高度纯化的靶细胞快速和简单的方法。
该议定书中的关键步骤
细胞活力和细胞产量
体外造血保持系细胞的活力，是确保成功的和可重复的实验，是至关重要的。化学和生物试剂，次优实验条件或切除器官的不适当…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究是由教育部，新加坡教育部（ACRF Tier1-RG40 / 13层2-MOE2013-T2-2-038）的支持。这份手稿是由来自Obrizus通信艾米沙利文编辑。
Materials
| Materials | |||
| RPMI 1640 (without L-Glutamine) | Gibco | 31870025 | |
| Fetal Bovine Serum | Heat inactivated | ||
| L-glutamine | Gibco | 25030024 | |
| Penicillin/Streptomycin | Gibco | 15140114 | |
| 2-mercaptoethanol | Gibco | 21985023 | |
| Anti-CD43 magnetic microbeads | Miltenyi Biotec | 130-049-801 | Mix well prior use |
| Streptavidin microbeads | Miltenyi Biotec | 130-048-101 | Mix well prior use |
| Anti-Annexin V magnetic beads | Miltenyi Biotec | 130-090-201 | Mix well prior use |
| MACS LD | Miltenyi Biotec | 130-042-901 | |
| 96-well U-bottom sterile culture plate | Greiner Bio-one | 650180 | |
| 96-well F-bottom sterile culture plate | Greiner Bio-one | 655180 | |
| 100 μm cell strainer mesh | To sterilize using UV radiation prior use | ||
| 0.2 μm sterile disposable filter units | Nalgene | 567-0020 | Can be substituted with any sterile filter device |
| CellTrace Violet | Invitrogen | C34557 | CTV for short; alternative to CFSE |
| CellTrace Yellow | Invitrogen | C34567 | CTY for short; alternative to CFSE |
| CellTrace Far Red | Invitrogen | C34564 | CTFR for short; alternative to CFSE |
| Cell Proliferation Dye eFluor 670 | eBioscience | 65-0840 | CPD670 for short; alternative to CFSE |
| PKH26 | Sigma Aldrich | PKH26GL | PKH26, alternative to CFSE |
| Name | Company | Catalog Number | Comments |
| Chemicals | |||
| Dextrose | Sigma Aldrich | G7021 | |
| Potassium phosphate monobasic | Sigma Aldrich | P5655 | |
| Sodium phosphate dibasic | Sigma Aldrich | S5136 | |
| Phenol Red | Sigma Aldrich | P0290 | |
| Calcium chloride dihydrate | Sigma Aldrich | C7902 | |
| Potassium chloride | Sigma Aldrich | P5405 | |
| Sodium chloride | Merck Millipore | S7653 | Can use from other sources |
| Magnesium chloride hexahydrate | Sigma Aldrich | M2393 | |
| Magnesium sulfate | Sigma Aldrich | M2643 | |
| Ammonium chloride | Sigma Aldrich | A9434 | |
| Tris-base | |||
| Dimethyl Sulfoxide | Sigma Aldrich | D8418 | |
| (5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) | Molecular Probes | C-1157 | Reconstitute in DMSO |
| Phorbol 12,13-dibutyrate (PBDU, Phorbol ester) | Sigma Aldrich | P1269 | |
| A23187 (Calcium ionophore) | Sigma Aldrich | C7522 | |
| Name | Company | Catalog Number | Comments |
| Antibodies and recombinant protein | |||
| CD11b biotin (clone m1/70) | Biolegend | 101204 | T cell depletion cocktail |
| CD11c biotin (clone N418) | Biolegend | 117304 | T cell depletion cocktail |
| Gr-1 biotin (clone RB6-8C5) | Biolegend | 108404 | T cell depletion cocktail |
| Ter119 biotin (clone Ter119) | Biolegend | 116204 | T cell depletion cocktail |
| TCR-γδ biotin (clone GL-3) | Biolegend | 118103 | T cell depletion cocktail |
| CD19 biotin (clone 6D5) | Biolegend | 115504 | T cell depletion cocktail |
| B220 biotin (clone RA3-6B2) | Biolegend | 103204 | T cell depletion cocktail |
| CD49b biotin (clone DX5) | Biolegend | 108904 | T cell depletion cocktail |
| CD4 biotin (clone GK1.5) | Biolegend | 100404 | T cell depletion cocktail |
| CD8 biotin (clone 53-6.7) | Biolegend | 100704 | T cell depletion cocktail |
| F(ab’)2 goat anti-mouse IgM (plate coated) | Jackson ImmunoResearch | 115-006-075 | 50 µl/well for coating (96-well) |
| Anti-mouse CD40 mAb (plate coated) | Pharmingen | 553722 | 50 µl/well for coating (96-well) |
| Recombinant IL-4 | ProSpec | Cyt-282 | |
| LPS from E. coli Serotype 055:B5 | Sigma Aldrich | L-4005 | |
| Anti-CD3 (clone clone OKT3) (plate coated) | eBioscience | 16-0037-85 | 50 µl/well for coating (96-well) |
| Anti-CD28 (clone clone 37.51 ) (plate coated) | eBioscience | 16-0281-85 | 50 µl/well for coating (96-well) |
| Recombinant IL-2 | ProSpec | Cyt-370 | |
| Albumin from chicken egg white, Ovalbumin | Sigma Aldrich | A7641 |
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