Это быстрый и всеобъемлющий метод иммунофенотипирования миелоидного Производные супрессоров (MDSC) и обогащению Gr-1<sup> +</sup> Лейкоцитов из селезенки мышей. Этот метод использует проточной цитометрии и сортировки клеток AutoMACS обогатить жизнеспособных Gr-1<sup> +</sup> Лейкоцитов до FACS сортировки MDSC для использования<em> В естественных условиях</em> И<em> В пробирке</em> Анализ.
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.
Это подробный метод для обработки и immunophentyping MDSC населения, которые применимы к различным лимфоидной ткани различных животных моделях. В частности, autoMACS обогащения может быть использован для выделения различных групп населения, включая лейкоцитарную Gr-1 истощения спленоцитов 4, о…
The authors have nothing to disclose.
Мы признаем цитометрии USF ядро потока фонда. Мы хотели бы поблагодарить д-ра Дениз Купер для совместного использования ресурсов. Мы также хотели бы поблагодарить майя Коэн, Лаура Пендлтон и Диана Латур за помощь в создании и съемках этого видео. Н.Н. поддержке NSF FG-LSAMP мост доктора стипендий HRD # 0929435. Эта работа финансировалась American Cancer Society институциональных исследований грант № 93-032-13/Moffitt онкологический центр награжден TG.
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 |