Name: preparation of myeloid derived suppressor cells (mdsc) from naive and pancreatic tumor-bearing mice using flow cytometry and automated magnetic activated cell sorting (automacs)
Uploaded: 2012-06-18T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell S

We acknowledge the USF Flow Cytometry Core Facility. We would like to thank Dr. Denise Cooper for sharing resources. We would also like to thank Maya Cohen, Laura Pendleton and Diana Latour for their assistance in setting up and filming of this video. NN supported by NSF FG-LSAMP Bridge to the Doctorate Fellowship HRD #0929435. This work was funded by the American Cancer Society Institutional Research Grant# 93-032-13/Moffitt Cancer Center awarded to TG.

Prior to starting, prepare the following solutions:
3% Staining Media (SM):
-3% Fetal Bovine Serum (FBS) in 1X Phosphate Buffer Saline (PBS)
MACS Buffer (MB):
- 0.5% Albumin from Bovine Serum (BSA) in 1XPBS
1. Harvest Spleens from Mice
<ol>
 <li>Subcutaneously inject 6-8 week of age C57BL/6 mice (Harlan) with 1.5 x 105 murine Panc02 cells s...

<ol>
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This is a detailed method for processing and immunophentyping MDSC populations that is applicable to different lymphoid tissues from various animal models. In particular, autoMACS enrichment can be used for the isolation of various leukocyte populations including Gr-1 depletion of splenocytes 4, purification of myeloid subsets from splenocytes and lymph nodes 5, isolation of bone marrow neutrophils 14 and purification of CD8+ T cells from spleen and lymph nodes 15. ...

<table border="1">
 <tbody>
 <tr>
 <td>REAGENT</td>
 <td>COMPANY</td>
 <td>CATALOG #</td>
 <td>COMMENTS</td>
 </tr>
 <tr>
 <td>1X Phosphate Buffered Saline</td>
 <td>Thermo Scientific Hyclone</td>
 <td>SH30028.02</td>
 <td>Ca2+/Mg2+/Phenol Red-free</td>
 </tr>
 <tr>
 <td>Albumin from Bovine Serum (BSA)</td>
 <td>Sigma-Aldrich</td>
 <td>A7906</td>
 <td>Let BSA dissolve undisturbed in PBS; Sterile Environment</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum (FBS)</td>
 <td>Thermo Scientific Hyclone</td>
 <td>SV3001403HI</td>
 <td>Heat Inactivated; Sterile Environment</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD16/32 monoclonal antibody (Fc Block)</td>
 <td>BD Biosciences</td>
 <td>553142</td>
 <td>Sterile Environment</td>
 </tr>
 <tr>
 <td>Anti-mouse CD11b (Mac-1) FITC </td>
 <td>eBiosciences</td>
 <td>11-0112</td>
 <td>Sterile Environment</td>
 </tr>
 <tr>
 <td>Anti-mouse Ly6G (Gr-1) APC </td>
 <td>eBiosciences</td>
 <td>17-5931</td>
 <td>Sterile Environment</td>
 </tr>
 <tr>
 <td>Anti-mouse Ly6G (Gr-1) PE</td>
 <td>eBiosciences</td>
 <td>12-5931</td>
 <td>Sterile Environment</td>
 </tr>
 <tr>
 <td>DAPI</td>
 <td>Invitrogen</td>
 <td>D1306</td>
 <td>Serial Dilution Sterile Environment</td>
 </tr>
 <tr>
 <td>Cell Dissociation Sieve</td>
 <td>Sigma-Aldrich</td>
 <td>CD1-1KT</td>
 <td>Autoclave before use</td>
 </tr>
 <tr>
 <td>70-&mu;m strainer</td>
 <td>BD Biosciences</td>
 <td>352350</td>
 <td>Sterile Environment</td>
 </tr>
 <tr>
 <td>1X RBC Lysis Buffer</td>
 <td>eBiosciences</td>
 <td>00-4333-57</td>
 <td>Warm to room temperature before use; Sterile Environment</td>
 </tr>
 <tr>
 <td>Petri dishes</td>
 <td>Fisher Scientific</td>
 <td>08-757-12</td>
 <td>Sterile Environment</td>
 </tr>
 <tr>
 <td>50ml conical tubes</td>
 <td>Thermo Scientific</td>
 <td>339652</td>
 <td>Sterile Environment</td>
 </tr>
 <tr>
 <td>5ml 12X75mm polystyrene round bottom tubes</td>
 <td>BD Biosciences</td>
 <td>352054</td>
 <td>Known as FACS tubes; Sterile Environment</td>
 </tr>
 <tr>
 <td>96-well V-bottom plates</td>
 <td>Corning</td>
 <td>3897</td>
 <td>Sterile Environment</td>
 </tr>
 <tr>
 <td>Trypan Blue</td>
 <td>Cellgro</td>
 <td>25-900-CI</td>
 <td>Sterile Environment</td>
 </tr>
 <tr>
 <td>PE MicroBeads</td>
 <td>Miltenyi Biotec</td>
 <td>130-048-801</td>
 <td>Sterile Environment</td>
 </tr>
 <tr>
 <td>AutoMACS Pro Separator</td>
 <td>Miltenyi Biotec</td>
 <td>130-092-545</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>AutoMACS Columns</td>
 <td>Miltenyi Biotec</td>
 <td>130-021-101</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>AutoMACS Running Buffer</td>
 <td>Miltenyi Biotec</td>
 <td>130-091-221</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

preparation of myeloid derived suppressor cells (mdsc) from naive and pancreatic tumor-bearing mice using flow cytometry and automated magnetic activated cell sorting (automacs)

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 na&iuml;ve 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 na&iuml;ve MDSC 13. MDSC percentages are significantly less in lymphoid compartments of na&iuml;ve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1+ leukocytes from na&iuml;ve 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 na&iuml;ve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas na&iuml;ve 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 na&iuml;ve 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 (na&iuml;ve vs. tumor- bearing) in in vivo and in vitro assays.

Watch this Scientific Journal Video about Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell S

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

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