अनुभवहीन और अग्नाशय के ट्यूमर असर चूहों से माइलॉयड व्युत्पन्न दबानेवाला कोशिकाओं (MDSC) फ्लो और स्वचालित चुंबकीय सक्रिय सेल छंटनी (AutoMACS) के का उपयोग कर की तैयारी
Summary
यह माइलॉयड व्युत्पन्न दबानेवाला कोशिकाओं (MDSC) immunophenotyping और जीआर एक समृद्ध की एक तेजी से और व्यापक विधि है<sup> +</sup> माउस spleens से leukocytes. इस पद्धति का प्रवाह cytometry और AutoMACS सेल लिए व्यवहार्य जीआर-1 के लिए समृद्ध छंटनी का उपयोग करता है<sup> +</sup> FACS के लिए पहले leukocytes उपयोग के लिए MDSC की छँटाई<em> Vivo में</em> और<em> इन विट्रो में</em> Assays के.
Abstract
MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer1-7. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens7. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to naïve counterparts7-10. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression11,12. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.
Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to naïve MDSC 13. MDSC percentages are significantly less in lymphoid compartments of naïve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1+ leukocytes from naïve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1+ leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1+ leukocytes from spleens of naïve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas naïve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from naïve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1+ leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1+ enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (naïve vs. tumor- bearing) in in vivo and in vitro assays.
Protocol
Discussion
यह प्रसंस्करण और immunophentyping MDSC आबादी है कि विभिन्न पशु मॉडल से अलग lymphoid ऊतकों को लागू करने के लिए एक विस्तृत विधि है. विशेष रूप में, autoMACS संवर्धन 4 splenocytes की जीआर-1 कमी, माइलॉयड सबसेट की splenocytes और लिम्फ नोड्स 5 से…
Disclosures
The authors have nothing to disclose.
Acknowledgements
हम USF के फ्लो Cytometry कोर सुविधा स्वीकार करते हैं. हम संसाधनों को साझा करने के लिए डॉ. डेनिस कूपर धन्यवाद देना चाहूंगा. हम भी उनकी सहायता के लिए स्थापित है और इस वीडियो की शूटिंग में माया कोहेन, लौरा Pendleton और डायना Latour का शुक्रिया अदा करना चाहते हैं. एनएन NSF के FG LSAMP ब्रिज से डॉक्टरेट फैलोशिप 0929435 मानव संसाधन विकास के लिए समर्थित है. इस काम अमेरिकन कैंसर सोसायटी संस्थागत अनुसंधान अनुदान # 93-032-13/Moffitt कैंसर टीजी के लिए सम्मानित किया गया केंद्र द्वारा वित्त पोषित किया गया था.
Materials
| REAGENT | COMPANY | CATALOG # | COMMENTS |
| 1X Phosphate Buffered Saline | Thermo Scientific Hyclone | SH30028.02 | Ca2+/Mg2+/Phenol Red-free |
| Albumin from Bovine Serum (BSA) | Sigma-Aldrich | A7906 | Let BSA dissolve undisturbed in PBS; Sterile Environment |
| Fetal Bovine Serum (FBS) | Thermo Scientific Hyclone | SV3001403HI | Heat Inactivated; Sterile Environment |
| Rat anti-mouse CD16/32 monoclonal antibody (Fc Block) | BD Biosciences | 553142 | Sterile Environment |
| Anti-mouse CD11b (Mac-1) FITC | eBiosciences | 11-0112 | Sterile Environment |
| Anti-mouse Ly6G (Gr-1) APC | eBiosciences | 17-5931 | Sterile Environment |
| Anti-mouse Ly6G (Gr-1) PE | eBiosciences | 12-5931 | Sterile Environment |
| DAPI | Invitrogen | D1306 | Serial Dilution Sterile Environment |
| Cell Dissociation Sieve | Sigma-Aldrich | CD1-1KT | Autoclave before use |
| 70-μm strainer | BD Biosciences | 352350 | Sterile Environment |
| 1X RBC Lysis Buffer | eBiosciences | 00-4333-57 | Warm to room temperature before use; Sterile Environment |
| Petri dishes | Fisher Scientific | 08-757-12 | Sterile Environment |
| 50ml conical tubes | Thermo Scientific | 339652 | Sterile Environment |
| 5ml 12X75mm polystyrene round bottom tubes | BD Biosciences | 352054 | Known as FACS tubes; Sterile Environment |
| 96-well V-bottom plates | Corning | 3897 | Sterile Environment |
| Trypan Blue | Cellgro | 25-900-CI | Sterile Environment |
| PE MicroBeads | Miltenyi Biotec | 130-048-801 | Sterile Environment |
| AutoMACS Pro Separator | Miltenyi Biotec | 130-092-545 | |
| AutoMACS Columns | Miltenyi Biotec | 130-021-101 | |
| AutoMACS Running Buffer | Miltenyi Biotec | 130-091-221 |
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