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 suspended in 100 &mu;l 1x PBS (tumor-bearing; TB). Control mice (Na&iuml;ve) receive 100 &mu;l 1XPBS.</li>
 <li>Approximately 4 weeks post injection, euthanize mice by carbon dioxide asphyxiation.</li>
 <li>Harvest spleens from mice by blunt dissection using forceps and scissors then weigh using a balance. Place spleens in separate, labeled 50 ml conical tubes containing 1 x PBS.</li>
</ol>
2. Generate a Single-cell Suspension of Leukocytes from Spleens
All procedures should be performed in a sterile environment under a Biological Safety Hood and cells and antibodies kept on ice.
<ol>
 <li>Assemble the cell dissociation sieve by inserting the mesh screen into the opening of the cup towards the bottom. Then, insert the retaining ring into the threaded area with the slotted side up and use the ring key to tighten the retaining ring, thus holding the screen in place. Place the assembled sieve in a Petri dish containing 10 ml 1 x PBS.</li>
 <li>Pool spleens and use glass pestle to grind spleens against the mesh screen of the cell dissociation sieve and into the Petri dish. Repeat for each treatment group of mice.</li>
 <li>Filter cell suspension into a 50 ml conical tube using a 70 &mu;m cell strainer and a 5 ml serological pipette. Centrifuge at 12,000 rpm (250-300 x g) for 5 min.</li>
 <li>Remove supernatant and resuspend pellet in 5 ml 1 x RBC lysis buffer per spleen. Pipet up and down vigorously. Incubate at room temperature for 5 min. Stop the reaction by adding 20 ml of 1 x PBS. Pipet up and down vigorously. Centrifuge at 12,000 rpm (250-300 x g) for 5 min.</li>
 <li>Remove supernatant and resuspend pellet in 20 ml sterile 1 x PBS. Pipet up and down vigorously.</li>
 <li>Count cells using trypan blue and a hemacytometer and resuspend at desired concentration in 3% SM so that 50 &mu;l is equivalent to 5x105-1x106 cells (1x107 cells/ml - 2x107 cells/ml).</li>
</ol>
3. Cell-surface Staining /Immunophenotyping of MDSC using Flow Cytometry
<ol>
 <li>Label the wells of a 96-well V bottom plate for control and experimental samples and single stains for compensation controls (No Stain, NS; Mac-1-FITC; Gr-1-APC and DAPI).</li>
 <li>Add 5x105-1x106 cells equivalent to 50 &mu;l/well of splenocytes to their respective wells in the 96-well V-bottom plate. Centrifuge plate at 12,000 rpm (250-300 x g) for 5 min.</li>
 <li>Prepare "Master Mix" (MM) of Mouse BD Fc Block (Rat anti-mouse CD16/32 monoclonal antibody) diluted in 3% SM, in a 1.5 ml microcentrifuge tube on ice. As a starting point, use 1 &mu;g Fc Block in 3% SM for a final volume of 50 &mu;l, per well.</li>
 <li>Carefully remove supernatant from each well of the 96-well V-bottom plate by quickly inverting the plate over and back, over a waste container or sink without disruption of the cell pellets.</li>
 <li>Vortex, briefly centrifuge Fc Block MM for 5 seconds and add 50 &mu;l to all pellets in the 96-well V-bottom plate. Mix well by gently pipetting up and down, leaving samples in their wells. Incubate plate for 15 min in the dark on ice. Centrifuge plate at 12,000 rpm (250-300 x g) for 5 min.</li>
 <li>Prepare "Master Mix" (MM) of fluorochrome-conjugated antibodies diluted in 3% SM in a 1.5ml microcentrifuge tube on ice, while samples incubate with Fc Block. Antibodies should be titrated to determine optimal dilutions for staining procedures. As a starting point, combine a 1:25 dilution of Mac-1-FITC and 1:20 dilution of Gr-1-APC in 3% SM for a final volume of 50 &mu;l, per sample well.</li>
 <li>Carefully remove supernatant from each well of the 96-well V -bottom as previously described.</li>
 <li>Vortex, briefly centrifuge MM of fluorescent-conjugated staining antibodies for 5 seconds and add 50 &mu;l to control and experimental pellets. Mix well by gently pipetting up and down. For single-stain compensations, add a 1:25 dilution of Mac-1-FITC, 1:20 dilution of Gr-1-APC and 75 ng/ml of DAPI, in 3% SM for a final volume of 50 &mu;l per well to their respective wells. Add 50 &mu;l 3% SM to unstained well (No Stain). Mix well and incubate cells in 96-well V-bottom plate for 30 min in the dark on ice.</li>
 <li>Label FACS tubes (5 ml, 12 mm x 75 mm polystyrene round bottom tubes) to correspond to each well in the 96-well V-bottom plate. Add 200 &mu;l 3% SM to each FACS tube.</li>
 <li>Centrifuge plate at 12,000 rpm (250-300 x g) for 5 min and remove supernatants. Wash pellets by adding 100 &mu;l 3% SM to each pellet and mix well by gently pipetting up and down. Centrifuge plate at 12,000 rpm (250-300 x g) for 5 min. Repeat wash step once more.</li>
 <li>Carefully remove supernatant from each well of the 96-well V-bottom as previously described. Resuspend each pellet in 100 &mu;l 3% SM and mix well.</li>
 <li>Transfer 100 &mu;l resuspended pellet from each well of the 96-well V-bottom plate to their respectively labeled FACS tube.</li>
 <li>Prior to Flow cytometry analysis, add 75 ng/ml DAPI to control and experimental samples and DAPI single stain compensation control.</li>
 <li>Perform flow cytometric data acquisition of MDSC percentages. Perform compensation using the negative (no stain control) and the single positive controls. Set up a dot plot that displays the forward (FSC) versus side scatter (SSC) in log scale so that leukocyte populations of interest can be identified. Draw a large gate on all leukocytes, excluding debris and clumps with lowest forward and side scatter. From this parent gate, create a new dot plot that displays SSC versus DAPI and gate on DAPI- (live) cells. Select this newly gated population and create a dot plot that displays Mac-1 versus Gr-1 and gate on your double positive (Mac-1+ Gr-1+) MDSC.</li>
</ol>
4. Magnetic Enrichment of Gr-1+ Leukocytes
<ol>
 <li>Aliquot 1x107 remaining unstained leukocytes into appropriately labeled FACS tubes and centrifuge at 12,000 rpm (250-300 x g) for 5 min.</li>
 <li>Prepare MM of Gr-1-PE antibody in a 1.5 ml microcentrifuge tube. For up to 107 cells, use a 1:10 dilution of Gr-1-PE antibody in 50 &mu;l MB, per sample. For greater cell numbers, scale up volumes accordingly. Briefly centrifuge for 5 seconds, add to leukocytes in FACS tubes and incubate for 15 min at 4 &deg;C in the dark.</li>
 <li>Add 2 ml of MB to FACS tubes, centrifuge at 12,000 rpm (250-300 x g) for 5 min and discard supernatant.</li>
 <li>Prepare MM of Anti-PE MicroBeads in a 1.5 ml microcentrifuge tube. For up to 107 cells, use a 1:4 dilution of anti-PE MicroBeads in 200 &mu;l MB, per sample. For greater cell numbers, scale up volumes accordingly. Briefly centrifuge for 5 seconds, add to leukocytes in FACS tubes and incubate for 15 min at 4 &deg;C in the dark.</li>
 <li>Add 2 ml of MB to FACS tubes, centrifuge at 12,000 rpm (250-300 x g) for 5 min and discard supernatant. Resuspend pellet in 3 ml of SB. Filter through a 70 &mu;m strainer into a new, labeled 50 ml conical tube.</li>
 <li>Prepare and prime Auto MACS Pro Separator. Refill all bottles with the appropriate solutions and empty the waste bottle, if necessary. Turn instrument on and examine status of fluid containers and column(s) after initialization. All symbols should be green. On the menu, select "Separation" from the upper menu bar followed by "Wash Now" from the lower menu bar. Select "Rinse" from the pop-up option followed by "Run" to start the priming process. Once the priming process is successfully completed, the instrument will then display that it is "Ready for Separation" under the Status menu.</li>
 <li>Choose suitable chilled tube rack for tube sizes and place 50 ml conical tube with magnetically labeled cells in row A, 50 ml conical tube for negative fraction collection in row B and 15 ml conical tube for positive fraction collection in row C.</li>
 <li>Choose "POSSEL_S" cell separation program for positive selection of labeled target cells in sensitive mode from samples. Magnetically labeled target cells are retained on the automats column; unlabeled cells are released into the negative fraction collection tube in row B. On automated retraction of the magnet, the labeled target cells will be released into the positive collection tube in row C of the tube rack.</li>
</ol>
5. Post-Sort Analysis of Gr-1+ Enriched Leukocytes
<ol>
 <li>Recount Gr-1+ and Gr-1- fractions using trypan blue and a hemacytometer. Resuspend cells at desired concentration in 3% staining medium so that 50 &mu;l is equivalent to 5x105-1x106 cells (1x107 cells/ml - 2x107 cells/ml) and transfer 50 &mu;l to correspondingly labeled FACS tubes.</li>
 <li>Prepare a MM of Mac-1 only, at a 1:25 dilution in 3% SM for a final volume of 50 &mu;l per sample. Stain cells and prepare single stain compensations of Gr-PE, Mac-1 FITC and DAPI for flow cytometry analysis. Add 200 &mu;l 3% SM and DAPI viability dye as previously described.</li>
 <li>Perform flow cytometric data acquisition to determine Gr-1+ and Gr-1- percentages and to also compare MDSC percentages pre- and post-autoMACS enrichment.</li>
</ol>
6. Representative Results
Here we show representative results for autoMACS enrichment of Gr-1+ leukocytes from pooled, na&iuml;ve spleens for subsequent FACS sorting of MDSC (Figure 1). 1x106 na&iuml;ve leukocytes were stained with Mac-1-FITC and Gr-1-APC antibodies to identify MDSC percentages using a BD LSRII instrument, prior to autoMACS sorting. 1x107 na&iuml;ve leukocytes were then stained with anti-Gr-1-PE antibodies and PE-MicroBeads for enrichment of Gr-1+ leukocytes using an autoMACS Pro Separator. Post-autoMACS enrichment, Gr-1 percentages in Gr-1+ and Gr-1- collected fractions were evaluated using flow cytometry. 1x106 Gr-1+ leukocytes were removed and stained with Mac-1-FITC antibody to analyze and compare MDSC percentages of enriched, pooled na&iuml;ve leukocytes to non-enriched, pooled tumor-bearing leukocytes by flow cytometry.
<img alt="Figure 1" src="/files/ftp_upload/3875/3875fig1.jpg" /> 
Figure 1. AutoMACS enrichment of Na&iuml;ve Gr-1+ leukocytes for MDSC FACS Sorting. Spleens were harvested from pancreatic tumor-bearing and na&iuml;ve mice and processed into single-cell suspensions. Flow cytometry analysis of na&iuml;ve leukocytes surface stained with Mac-1 and Gr-1 fluorescent-conjugated antibodies, prior to autoMACS enrichment (A). Flow cytometry analysis of Gr-1+ (B) and Gr-1- (C) fractions post- autoMACS enrichment of Gr-1+ cells from pooled na&iuml;ve leuckocytes stained with Gr-1-PE antibodies and anti-PE MicroBeads. Flow cytometry analysis of MDSC and Gr-1+ percentages post-autoMACS enrichment of Gr-1+ cells from pooled na&iuml;ve leukocytes (D) compared to non-enriched pooled leukocytes from tumor-bearing mice (E) (na&iuml;ve mice, n=5; tumor-bearing mice, n=3). MDSC and Gr-1 percentages are gated in the representative contour plots and histograms.

<ol>
	<li>Goni, O., Alcaide, P., Fresno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosuppression+during+acute+Trypanosoma+cruzi+infection:+involvement+of+Ly6G+(Gr1(+))CD11b(+)+immature+myeloid+suppressor+cells.">Immunosuppression during acute Trypanosoma cruzi infection: involvement of Ly6G (Gr1(+))CD11b(+) immature myeloid suppressor cells.</a> Int. Immunol. 14, 1125-1134 (2002).</li><li>Zhu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD11b+Ly-6C(hi)+suppressive+monocytes+in+experimental+autoimmune+encephalomyelitis.">CD11b+Ly-6C(hi) suppressive monocytes in experimental autoimmune encephalomyelitis.</a> J. Immunol. 179, 5228-5237 (2007).</li><li>Makarenkova, V. P., Bansal, V., Matta, B. M., Perez, L. A., Ochoa, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD11b+/Gr-1++myeloid+suppressor+cells+cause+T+cell+dysfunction+after+traumatic+stress.">CD11b+/Gr-1+ myeloid suppressor cells cause T cell dysfunction after traumatic stress.</a> J. Immunol. 176, 2085-2094 (2006).</li><li>Ghansah, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+myeloid+suppressor+cells+in+SHIP-deficient+mice+represses+allogeneic+T+cell+responses.">Expansion of myeloid suppressor cells in SHIP-deficient mice represses allogeneic T cell responses.</a> J. Immunol. 173, 7324-7330 (2004).</li><li>Paraiso, K. H., Ghansah, T., Costello, A., Engelman, R. W., Kerr, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+SHIP+deficiency+expands+myeloid+regulatory+cells+and+abrogates+graft-versus-host+disease.">Induced SHIP deficiency expands myeloid regulatory cells and abrogates graft-versus-host disease.</a> J. Immunol. 178, 2893-2900 (2007).</li><li>Yin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid-derived+suppressor+cells+prevent+type+1+diabetes+in+murine+models.">Myeloid-derived suppressor cells prevent type 1 diabetes in murine models.</a> J. Immunol. 185, 5828-5834 (2010).</li><li>Gabrilovich, D. I., Nagaraj, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid-derived+suppressor+cells+as+regulators+of+the+immune+system.">Myeloid-derived suppressor cells as regulators of the immune system.</a> Nat. Rev. Immunol. 9, 162-174 (2009).</li><li>Gallina, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumors+induce+a+subset+of+inflammatory+monocytes+with+immunosuppressive+activity+on+CD8++T+cells.">Tumors induce a subset of inflammatory monocytes with immunosuppressive activity on CD8+ T cells.</a> J. Clin. Invest. 116, 2777-2790 (2006).</li><li>Zhao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+frequency+of+myeloid-derived+suppressor+cells+in+mice+with+spontaneous+pancreatic+carcinoma.">Increase in frequency of myeloid-derived suppressor cells in mice with spontaneous pancreatic carcinoma.</a> Immunology. 128, 141-149 (2009).</li><li>Greten, T. F., Manns, M. P., Korangy, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+derived+suppressor+cells+in+human+diseases.">Myeloid derived suppressor cells in human diseases.</a> Int Immunopharmacol. 11, 802-806 (2011).</li><li>Youn, J. I., Nagaraj, S., Collazo, M., Gabrilovich, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subsets+of+myeloid-derived+suppressor+cells+in+tumor-bearing+mice.">Subsets of myeloid-derived suppressor cells in tumor-bearing mice.</a> J. Immunol. 181, 5791-5802 (2008).</li><li>Ribechini, E., Greifenberg, V., Sandwick, S., Lutz, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subsets,+expansion+and+activation+of+myeloid-derived+suppressor+cells.">Subsets, expansion and activation of myeloid-derived suppressor cells.</a> Med. Microbiol. Immunol. 199, 273-281 (2010).</li><li>Pilon-Thomas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+Pancreatic+Adenocarcinoma+Dampens+SHIP-1+Expression+and+Alters+MDSC+Homeostasis+and+Function.">Murine Pancreatic Adenocarcinoma Dampens SHIP-1 Expression and Alters MDSC Homeostasis and Function.</a> PLoS One. 6, (2011).</li><li>Panopoulos, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT3+governs+distinct+pathways+in+emergency+granulopoiesis+and+mature+neutrophils.">STAT3 governs distinct pathways in emergency granulopoiesis and mature neutrophils.</a> Blood. 108, 3682-3690 (2006).</li><li>Preynat-Seauve, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extralymphatic+tumors+prepare+draining+lymph+nodes+to+invasion+via+a+T-cell+cross-tolerance+process.">Extralymphatic tumors prepare draining lymph nodes to invasion via a T-cell cross-tolerance process.</a> Cancer Res. 67, 5009-5016 (2007).</li><li>Davies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separations+by+flow+cytometry.">Cell separations by flow cytometry.</a> Methods Mol. Med. 58, 3-15 (2001).</li><li>Maecker, H., Trotter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selecting+reagents+for+multicolor+BD+flow+cytometry.">Selecting reagents for multicolor BD flow cytometry.</a> Postepy Biochem. 55, 461-467 (2009).</li><li>Bagwell, C. B., Adams, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Spectral+Overlap+Compensation+for+Any+Number+of+Flow+Cytometry+Parameters.">Fluorescence Spectral Overlap Compensation for Any Number of Flow Cytometry Parameters.</a> Annals of the New York Academy of Sciences. 677, 167-184 (1993).</li><li>Perfetto, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amine+reactive+dyes:+an+effective+tool+to+discriminate+live+and+dead+cells+in+polychromatic+flow+cytometry.">Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry.</a> J. Immunol. Methods. 313, 199-208 (2006).</li><li>Safarik, I., Safarikova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+magnetic+techniques+for+the+isolation+of+cells.">Use of magnetic techniques for the isolation of cells.</a> J. Chromatogr. B. Biomed. Sci. Appl. 722, 33-53 (1999).</li><li>Collazo, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHIP+limits+immunoregulatory+capacity+in+the+T-cell+compartment.">SHIP limits immunoregulatory capacity in the T-cell compartment.</a> Blood. 113, 2934-2944 (2009).</li><li>Mack, E., Neubauer, A., Brendel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+RNA+yield+from+small+cell+populations+sorted+by+flow+cytometry+applying+different+isolation+procedures.">Comparison of RNA yield from small cell populations sorted by flow cytometry applying different isolation procedures.</a> Cytometry. A. 71, 404-409 (2007).</li><li>Strauss, L., Czystowska, M., Szajnik, M., Mandapathil, M., Whiteside, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+responses+of+human+regulatory+T+cells+(Treg)+and+effector+T+cells+to+rapamycin.">Differential responses of human regulatory T cells (Treg) and effector T cells to rapamycin.</a> PLoS One. 4, e5994 (2009).</li></ol>

No conflicts of interest declared.

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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26.1K Views.  University of South Florida Morsani College of Medicine. 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.

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

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. Determining the real time state of a microbial cell goes beyond live or dead categories, as microbes can exist in a dormant state, whereby cell division and metabolic activities are reduced. Given the need for detection and quantification of microbes, flow cytometry (FCM) with molecular probes provides a rapid and accurate method to help determine overall population viability. By using SYTOX Green and SYTOX Orange in the model cyanobacteria Microcystis aeruginosa to detect membrane integrity, we develop a transferable method for rapid indication of single cell mortality. The molecular probes used within this journal will be referred to as green or orange nucleic acid probes respectively (although there are other products with similar excitation and emission wavelengths that have a comparable modes of action, we specifically refer to the fore mentioned probes). Protocols using molecular probes vary between species, differing principally in concentration and incubation times. Following this protocol set out on M.aeruginosa the green nucleic acid probe was optimized at concentrations of 0.5 &#181;M after 30 min of incubation and the orange nucleic acid probe at 1 &#181;M after 10 min. In both probes concentrations less than the stated optimal led to an under reporting of cells with membrane damage. Conversely, 5 &#181;M concentrations and higher in both probes exhibited a type of non-specific staining, whereby &#39;live&#39; cells produced a target fluorescence, leading to an over representation of &#39;non-viable&#39; cell numbers. The positive controls (heat-killed) provided testable dead biomass, although the appropriateness of control generation remains subject to debate. By demonstrating a logical sequence of steps for optimizing the green and orange nucleic acid probes we demonstrate how to create a protocol that can be used to analyse cyanobacterial physiological state effectively.

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

Microbial populations contain substantial cell heterogeneity, which can dictate overall behavior. Molecular probe analysis through flow cytometry can determine physiological states of cells, however its application varies between species. This study provides a protocol to accurately determine cell mortality within a cyanobacterium population, without underestimating or recording false positive results.

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.