Applications for the MACSQuant Analyzer *

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1 For research use only Enumeration of CD34/CD133 positive cells with the CD34/CD133 Enumeration Kit Applications for the MACSQuant Analyzer * Background The CD34 antigen is a single-chain transmembrane glycoprotein, expressed on human hematopoietic stem and progenitor cells constituting a small subpopulation of bone marrow cells and peripheral blood cells. The antigen is absent on fully differentiated hematopoietic cells, such as normal peripheral blood lymphocytes, monocytes, granulocytes, erythrocytes, and platelets. After severe damage, for example, after myeloablative conditioning, the hematopoietic system can be reconstituted by transplantation of allogenic or autologous CD34 + hematopoietic progenitor cells (HPC). In the CD34/CD133 Enumeration Kit the antibody clone AC136 recognizes a class III epitope of the CD34 antigen. CD133 is a 5-transmembrane cell surface antigen, expressed on a subset of CD34 bright stem and progenitor cells in human fetal liver, bone marrow, cord blood, and peripheral blood but is not found on mature blood cells.¹ In contrast to the CD34 antigen, CD133 is not expressed by late progenitors, such as pre-b-cells, CFU-E, and CFU-G.², ³ CD133 has also been found to be expressed on circulating endothelial progenitor cells and fetal neural stem cells as well as on other tissue-specific stem cells, such as renal, prostate, and corneal stem cells.⁴ ⁶ In the CD34/CD133 Enumeration Kit the CD133/2 (clone 239C3) antibody recognizes epitope 2 of the CD133 antigen. CD34 + and CD34 + CD133 + cells may have various therapeutic uses. CD34 and CD133 enriched stem cell grafts have been employed in autologous⁷ ⁸ and allogenic transplantation both in the haploidentical⁹ ¹¹ as well as the HLA-matched setting¹². The high potential for hematopoietic engraftment of isolated CD133 + cells has been shown in NOD/SCID repopulation assays.¹³,¹⁴ CD34 and CD133 enriched cell fractions have also been used as starting fraction for ex vivo expansion of hematopoietic progenitor cells from cord blood.¹⁵ ¹⁸ Moreover CD133 + stem cells can be utilized in nonhematological applications, such as regenerative medicine. These cells have been shown to harbor the capability to differentiate into cell types of various tissues, for example, endothelial cells, neural cells, and hepatocytes. Therefore, CD133 + stem cells have come into focus in nonhematological applications especially in ischemic heart diseases.¹⁹ ²⁶ CD34 + /CD133 + HPC enumeration using the MACSQuant Analyzer The CD34/CD133 HPC Enumeration Kit is designed for the accurate detection of CD34 + /CD133 + HPCs from human blood samples by flow cytometry. The kit contains all of the necessary stainings for CD34/CD133 detection as well as controls for CD133 + HPC enumeration and viability. The gating strategy of the CD34/CD133 Enumeration Kit is based on the ISHAGE guidelines²⁷ for simple and standardized enumeration of CD34 + cells. By employing anti-, anti-, and anti- CD133/2-PE the kit allows the identification of CD45 + leucocytes, CD34 + HPCs, and CD34/CD133 HPCs. CD34 + as well as CD34 + /CD133 + HPCs are characterized by a dim CD45 + fluorescence and a low side scatter. The kit is designed for single platform technique²⁸, enabling absolute cell count determination with the MACSQuant Analyzer without the need to add further reagents, for example, counting beads. Dead cells can be excluded from the analysis by addition of the DNA stain 7-aminoactinomycin D (7-AAD), which is also included in the kit. 7-AAD diffuses through the cell membrane of dead cells and intercalates with their DNA. Materials and Methods Reagents and Solutions CD34/CD133 Enumeration Kit (# , for in vitro diagnostic use) Cell sample Double-distilled water * Applications on the MACSQuant Analyzer, MACSQuant Analyzer 1, and MACSQuant VYB are for research use only. Applications for the MACSQuant Analyzer June Copyright 213 Miltenyi Biotec GmbH. All rights reserved.

2 Buffer for optional dilution steps: Prepare a PEB (PBS/ EDTA/BSA) buffer containing phosphate-buffered saline (PBS), ph 7.2,.5% bovine serum albumin (BSA), and 2 mm EDTA, e.g., by diluting MACS BSA Stock Solution (# ) 1:2 with automacs Rinsing Solution (# ). Keep buffer cold (2 8 C). Note: For one test at least 2 µl of cell sample are required (analysis sample and control sample). We recommend to stain the analysis sample in dublicates to gain statistically relevant results. Materials MACSQuant Analyzer Micropipettes with disposable tips: variable micropipettes with volume ranges of 1 1 μl and 1 1 μl Vortex mixer Disposable capped polystyrene tubes mm for use whith MACSQuant Analyzer Sample preparation The following protocol can be used for CD34/CD133 HPC enumeration from various human blood products, such as whole blood, leukapheresis harvest, bone marrow, cord blood, and all fractions of CD34 + and CD133 + cells separated using a CliniMACS System. Staining of CD34 + /CD133 + cells Please refer to the package insert of the CD34/CD133 Enumeration Kit for sample requirements and preparation of solutions. 1. For each sample label two mm tubes with A (CD133 Control) and B (CD34/CD133 Staining Cocktail). 2. Add 2 μl of CD133 Control into tube A and 2 μl of CD34/CD133 Staining Cocktail into tube B. 3. Add 2 μl of 7-AAD solution into both tubes. 4. Carefully pipette 1 μl of well-mixed cell sample to the bottom of each labelled tube. Use reverse pipetting for all pipetting steps with blood products or RBC Lysis Solution (see package insert of the CD34/CD133 Enumeration Kit chapter 6). Immediately vortex thoroughly for 3 seconds and incubate for 1 minutes at 2 8 C in the dark. 5. Add 186 μl of 1 RBC Lysis Solution to each tube. Immediately vortex thoroughly for 3 seconds and incubate for 1 minutes at room temperature in the dark. 6. The analysis should be performed within one hour after staining. 7. Store the samples on ice until analysis. Staining summary Flow cytometric data acquisition and analysis with the MACSQuant Analyzer Note: For detailed information on operating the MACSQuant Instrument, please refer to the MACSQuant Instrument user manual and the MACSQuantify TM Software guide. 1. The analysis is performed with the single tube holder. Make sure the single tube holder is attached to the instrument and select single tube holder from the Rack drop down menu. 2. Load the appropriate instrument settings for the cell type by clicking Open, Instrument Settings, and select the correct instrument settings with compensation. 3. Open two analysis windows, the first one containing nine dot plots and the second one containing at least one large statistics display. Define the plot windows as follows: A Plot: versus side scatter B Plot: versus side scatter C Plot: 7-AAD versus side scatter D Plot: versus side scatter E Plot: versus side scatter F Plot: versus side scatter G Plot: CD133/2-PE versus H Plot: versus side scatter I Plot: versus side scatter 4. Set the trigger on the channel (B1) during data acquisition. Choose Edit, Instrument Settings, and select the trigger on the FITC-Channel (B1). Note: CD34 + and CD34 + /CD133 + progenitor cells express CD45 with lower staining intensity than lymphocytes. Be careful when setting the threshold and do not exclude these events from the analysis. Figure 1: Setting the threshold on the channel by selecting the trigger on the FITC-channel B1. Sample type A: CD133 control B: CD34/ CD133 Staining cocktail CD 133 Control [μl] CD34/133 Staining Cocktail [μl] 7-AAD [μl] Specimen [μl] Applications for the MACSQuant Analyzer June Copyright 213 Miltenyi Biotec GmbH. All rights reserved.

3 5. Acquire samples with height parameter activated which can be chosen in the instrument settings. Press Edit, Instrument Settings, Advanced, and mark Height. Note: It is important to gate the staining in Height (-H). It does not matter whether the other channels are gated on Height (...-H) or Area (...-A). Gating overview Gate Label Definition P1 Not P1 P2 WBCs Not P1/P2 P3 Viable WBCs Not P1/P2/P3 P4 Not P1/P2/P3/P4 P5 Cluster control CD34 Not P1/P2/P3/P4/P5 P6 CD34 + HPCs Not P1/P2/P3/P4/P5/P6 P7 P8 Cluster control CD133 Not P1/P2/P3/P4/P5/P6/P7 Not P1/P2/P3/P4/P5/P6/P7/P8 P9 CD133 + HPCs Not P1/P2/P3/P4/P5/P6/P7/P8/P9 Figure 2: The parameter Height is activated in the instrument settings. Note: At least 1. CD45 positive events per tube and a minimum of 1 CD133 positive events should be counted if possible. When analyzing a target cell fraction of a CliniMACS Separation at least 1, CD45 positive events should be counted. Note: All samples should be acquired in high -flowrate if possible. A Plot: (FSC) versus side scatter (SSC) Activated gate: no gate Define region P1 in this plot. P1 includes the SSC low and FSC low events. The events inside this region should be excluded from the further analysis by defining region P1 as an exclusion gate ( not-region ). 1. Choose the Samples tab 2. Mark the relevant file and open it by clicking on + 3. Choose region P1 and click right on it. By pressing Region Properties and marking Not the region becomes a not-region Data acquisition and analysis Analyze the samples in the following order: 1. CD133 control (tube A) 2. CD34/CD133 Staining cocktail (tube B) Recommended acquisition volumes for different sample types on MACSQuant Analyzer: Sample Whole blood Leukapheresis harvest Bone marrow Cord blood CliniMACS CD34 + target cell fraction CliniMACS CD133 + target cell fraction Recommended aquisition volumes 45 μl 2 45 μl 2 45 μl 3 45 μl 3 45 μl 3 45 μl Figure 3: Defining P1 as a not-region. Description of the detailed gating strategy The detailed description of the gating strategy is shown using a cord blood sample. The regions P1 P7 in plot A to G are defined during acquisition of the CD133 control sample. The regions P8 and P9 in plot H and I are defined during acquisition of the enumeration sample. B Plot: -H versus side scatter Activated gate: not P1 Set P2 to exclude all CD45 negative events from the analysis while all CD45 positive cells are included. Note: CD34 + and CD34 + /CD133 + progenitor cells express CD45 with lower staining intensity than lymphocytes. Applications for the MACSQuant Analyzer June Copyright 213 Miltenyi Biotec GmbH. All rights reserved.

4 Be careful when setting region P2 and do not exclude these events from the analysis ¹ 1² 1³ F Plot: CD34 + HPC determination plot; forward scatter versus side scatter Activated gate: not P1/P2/P3/P4/P5 Define a region P6 that identifies a cluster of events meeting all the fluorescence and light scatter criteria of CD34 positive progenitor cells. Cells clustered in region P6 exhibit slightly higher forward scatter than that of small lymphocytes and uniformly low side scatter. Any events outside region P6 are not included in the % viable CD34 positive cells determination. The proper setting of this region should be checked by showing this region in the first dot plot (A). The region has to enclose all lymphocytes. C Plot: 7-AAD versus side scatter Activated gate: not P1/P2 = WBCs Define P3 to include all 7-AAD negative viable WBCs ¹ 1² 7-AAD D Plot: versus side scatter Activated gate: not P1/P2/P3 = viable WBC Define P4 to include all CD34 positive cells with a low SSC ¹ 1² E Plot: CD34 cluster control plot: -H versus side scatter Activated gate: not P1/P2/P3/P4 The CD34 + cells form a cluster with low SSC and dim CD45 fluorescence and are gated in region P5. Non-specifically stained events are excluded from this region. 1 1³ 1³ G Plot: CD133/2-PE versus Activated gate: not P1/P2/P3/P4/P5/P6 Define P7 with the CD133 Control (A) to exclude all CD133 negative events. It is important to set region P7 as close as possible to the CD133 negative events to include all CD133 positive and CD133dim-positive cells. A maximum of 1 events is allowed in region P7 on the CD133 control sample. 1³ 1² 1¹ CD133 Control CD133/2-PE ¹ 1² 1³ 1 1¹ 1² 1³ 1³ 1² 1¹ CD34/CD133 Staining cocktail CD133/2-PE After defining plots A to G with tube A, apply all gates to tube B, by activating the A button and using the next sample button to load the data from tube B. For more information, please refer to the MACSQuantify Software guide ¹ 1² 1³ Applications for the MACSQuant Analyzer June Copyright 213 Miltenyi Biotec GmbH. All rights reserved.

5 H Plot: CD133 + cluster control plot: -H versus side scatter Activated gate: not P1/P2/P3/P4/P5/P6/P7 The CD34 + /CD133 + cells form a cluster with low SSC and dim CD45 fluorescence and are gated in region P8. Nonspecifically stained events are excluded from this region I Plot: CD133 + HPC determination plot; forward scatter versus side scatter Activated gate: not P1/P2/P3/P4/P5/P6/P7/P8 Define a region P9 that identifies a cluster of events meeting all the fluorescence and light scatter criteria of CD34 + /CD133 + progenitor cells. Cells clustered in region P9 exhibit slightly higher forward scatter than that of small lymphocytes and uniformly low side scatter. Any events falling outside region P9 are not included in the % viable CD34 + /CD133 + cells determination. The proper setting of this region should be checked by showing this region in the first dot plot (A). The region has to enclose all lymphocytes. 1 1¹ 1² 1³ Overview of the complete analyis window containing the dot plots ¹ 1² 1³ 1 1¹ 1² 1³ 7-AAD ¹ 1² 1³ 1 1¹ 1² 1³ 1³ 1 1² 1¹ ¹ 1² 1³ 1 1¹ 1² 1³ CD133/2-PE Applications for the MACSQuant Analyzer June Copyright 213 Miltenyi Biotec GmbH. All rights reserved.

6 Region %-# Count Count/ml e5 P e5 P e5 P3 (A) (E) (B) 7.44e5 P e3 P e3 P6 1. (D) 433 (C) 2.16e3 P e3 P e3 P9 1. (G) 365 (F) 1.82e3 Analysis of results using the MACSQuant Analyzer For analysis of results display a statistic table on page two of the analysis with the following parameters: count, count/ml, and percentage of superior gate (%#). The highlighted values are required for calculations. The dilution factor is calculated by multiplying an optional pre-dilution factor of the sample (no pre-dilution: factor = 1) by 2 μl divided by the sample volume used for staining (1 μl). Dilution factor in standard application without predilution = 2. Dilution factor = Pre-dilution factor 2 μl/sample volume A: The viability of WBC can be adopted from the statistic from P3 %#. B: The viable WBC [cells/ml] can be derived by multiplying the table item count/ml P3 with the dilution factor (2). Example of a calculation according to the statistic table (values of actual cord blood samples shown in dot plots A-I) Dilution Factor = 1 2 μl/1 μl = 2 A: Viability of WBC = % B: Viable WBC [count/ml] = ⁵ 2 (dilution factor) = ⁷ C: Viable CD34 + HPC [count/ml] = ³ 2 (dilution factor) = ⁴ D+E: % viable CD34 + HPC = 433 1/14911 =.29 % F: Viable CD34 + /CD133 + HPC count/ml = ³ 2 (dilution factor) = ⁴ G: % viablecd34 + /CD133 + HPC = 365 1/14911 =.24 % Viable WBC [cells/ml] = Count/mL P3 Dilution factor C: The viable CD34 + HPC [cells/ml] can be derived by multiplying the table item count/ml P6 with the dilution factor (2). Viable CD34 + HPC [cells/ml] = Count/mL P6 Dilution factor D+E: The percentage of viable CD34 + HPC can be calculated by multiplying the table item count P6 (D) by 1 and dividing this by the table item Count P3 (E). % viable CD34 + HPC = Count P6 1 Count P3 F: The viable CD34 + /CD133 + HPC [cells/ml] can be derived by multiplying the table item count/ml P9 with the dilution factor (2). Viable CD34 + /CD133 + HPC [cells/ml] = Count/mL P9 Dilution factor G: The percentage of viable CD34 + /CD133 + HPC can be calculated by multiplying the table item count P9 (G) by 1 and dividing this by the table item Count P3 (E). % viable CD34 + /CD133 + HPC = Count P9 1 Count P3 Applications for the MACSQuant Analyzer June Copyright 213 Miltenyi Biotec GmbH. All rights reserved.

7 References 1. Yin, A. H. et al. (1997) AC133, a Novel Marker for Human Hematopoietic Stem and Progenitor Cells. Blood 9: Freund, D. et al. (26) Comparative analysis of proliferative potential and clonogenicity of MACS-immunomagnetic isolated CD34 + and CD133 + blood stem cells derived from a single donor. Cell Prolif. 39: Takahiro, S., et al. (26) Highly Efficient Ex Vivo Expansion of Human Hematopoietic Stem Cells Using Delta1-Fc Chimeric Protein. Stem Cells 24: Gehling, U. et al. (2) In vitro differentiation of endothelial cells from AC133- positive progenitor cells. Blood 95: Peichev, M. et al. (2) Expression of VEGFR-2 and AC133 by circulating human CD34 + cells identifies a population of functional endothelial precursors. Blood 95: Uchida, N. et al. (2) Direct isolation of human central nervous system stem cells. Proc. Natl. Acad. Sci. USA 97: Koehl, U. et al. (22) Autologous transplantation of CD133 selected hematopoietic progenitor cells in a pediatric patient with relapsed leukemia. Bone Marrow Transplant. 29: Feller, N. et al. (25) Immunologic Purging of Autologous Peripheral Blood Stem Cell Products Based on CD34 and CD133 Expression Can Be Effectively and Safely Applied in Half of the Acute Myeloid Leukemia Patients. Clin. Cancer Res. 11: Lang, P. et al. (24) Transplantation of a combination of CD133 + and CD34 + selected progenitor cells from alternative donors. Br. J. Haematol. 124: Lang, P. et al. (24) Correction of persistent thrombocytopenia by a boost of CD133 + selected stem cells in a patient transplanted for Wiskott-Aldrich syndrome 1 years ago. Bone Marrow Transplant. 33: Bitan, M. et al. (25) Successful transplantation of haploidentically mismatched peripheral blood stem cells using CD purified stem cells. Exp. Hematol. 33: Bornhäuser, M. et al. (25) Rapid reconstitution of dendritic cells after allogeneic transplantation of CD133 + selected hematopoietic stem cells. Leukemia 19: Kuci, S. et al. (23) Identification of a novel class of human adherent CD34- stem cells that give rise to SCID-repopulating cells. Blood 11: Handgretinger, R. et al. (23) Biology and plasticity of CD133 + hematopoietic stem cells. Ann. N.Y. Acad. Sci. 996: Pasino, M. et al. (2) Flow cytometric and functional characterization of AC133 + cells from human umbilical cord blood. Br. J. Haematol. 18: Forraz, N. et al. (22) AC133 + umbilical cord blood progenitors demonstrate rapid self-renewal and low apoptosis. Br. J. Haematol. 119: Kobari, L. et al. (21) CD133 + cell selection is an alternative to CD34 + cell selection for ex vivo expansion of hematopoietric stem cells. J. Hematother Stem Cell Res. 1: Peled, T. et al. (24) Pre-clinical development of cord blood derived progenitor cell graft expanded ex vivo with cytokines and the polyamine copper chelator tetraethylenepentamine. Cytotherapy 6: Pompilio, G. et al. (24) Autologous peripheral blood stem cell transplantation for myocardial regeneration: a novel strategy for cell collection and surgical injection. Ann. Thorac. Surg. 78: Klein, H.M. et al. (24) Autologous bone marrow-derived stem cell therapy in combination with TMLR. A novel therapeutic option for endstage coronary heart disease: report on 2 cases. Heart Surg. Forum 7: E416 E Ghodsizad, A. et al. (24) Intraoperative isolation and processing of BM-derived stem cells. Cytother. 6: Stamm, C. et al. (24) CABG and bone marrow stem cell transplantation after myocardial infarction. Thorac. Cardiovasc. Surg. 52: Stamm, C. et al. (23) Autologous bone-marrow stem-cell transplantation for myocardial regeneration. Lancet 361: Lambiase, P.D. et al. (24) Circulating humoral factors and endothelial progenitor cells in patients with differing coronary colateral support. Circulation 19: Burt, R. et al. (23) Hematopoietic stem cell transplantation for cardiac and peripheral vascular disease. Bome Marrow Transplant. 32 (Suppl. 1): S29 S Pompilio, G. et al. (25) Long-lasting improvement of myocardial perfusion and chronic refractory angina after autologous intramyocardial PBSC transplantation. Cytotherapy 7: Sutherland, D.R. et al. (1996) The ISHAGE guidelines for CD34 + cell determination by flow cytometry. International Society of Hematotherapy and Graft Engineering. J. Hematotherapy 5 (3): Keeney, M. et al. (1998) Single platform flow cytometric absolute CD34 + cell counts based on the ISHAGE guidelines. International Society of Hematotherapy and Graft Engineering. Cytometry 34 (2): 61 7 Miltenyi Biotec provides products and services worldwide. Visit to find your nearest Miltenyi Biotec contact. Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. CliniMACS, MACS and MACSQuant are a registered trademarks of Miltenyi Biotec GmbH. Copyright 213 Miltenyi Biotec GmbH. All rights reserved. Applications for the MACSQuant Analyzer June Copyright 213 Miltenyi Biotec GmbH. All rights reserved.