140-002-274.04 1. 1.1 1. Description 4. This protocol describes the preparation of CD146 preparation the describes protocol This 2.1 1.1 Contents 2. Capacity Components 3. CD146 MicroBead Kit. CD146 MicroBead digestion, the centrifuged cell pellet, termed the stromal vascular vascular stromal the termed pellet, cell centrifuged the digestion, Product format Briefly, the lipoaspirate is first washed thoroughly in phosphate- in thoroughly washed first is lipoaspirate the Briefly, using collagenase in order to obtain a single-cell suspension. After After suspension. asingle-cell obtain to order in collagenase using fraction (SVF), is resuspended in buffer before serial filtration filtration serial before buffer in (SVF), resuspended is fraction macsde www.miltenyibiotec.com www.miltenyibiotec.com Storage 100 through surgery. cosmetic from obtained lipoaspirate or abdomen thigh is then counted and cells are then ready for separation using the the using for separation ready then are cells and counted then is buffered saline (PBS) before being subjected to enzymatic digestion digestion enzymatic to subjected (PBS) being before saline buffered Phone Phone Friedrich-Ebert-Straße Friedrich-Ebert-Straße Miltenyi Biotec B.V. KG &Co. Biotec Miltenyi

Protocol Description (SVF) from human lipoaspirate human from (SVF) References Example of a separation using the CD146 MicroBead Kit CD146 the MicroBead using of aseparation Example Principle of the preparation of the stromal vascular fraction fraction vascular stromal of the preparation of the Principle

2.3 2.2 1.5 1.4 Applications 1.3 1.2 +49 2204 8306-0, 2204 +49 @

miltenyi.com

from human lipoaspirate human from lipoaspirate human from (SVF) fraction Magnetic separation Magnetic labeling Magnetic (SVF) fraction vascular stromal of the Preparation requirements instrument and Reagent information Background Separation MACS® of the Principle vascular stromal of the preparation of the Principle 40 then and μm

For 10⁹ total cells, up to 100 separations. up100 to cells, For 10⁹ total Store protected from at 2−8 light from Store protected Human IgG. Human 2 mL FcR Blocking Reagent, human: Reagent, Blocking FcR 2 mL human: CD1462 mL MicroBeads, CD146 MicroBeads are supplied in buffer buffer in supplied are CD146 MicroBeads vial label. vial MicroBeads conjugated to monoclonal anti- monoclonal to conjugated MicroBeads containing stabilizer and 0.05% sodium azide. 0.05% sodium and stabilizer containing freeze. The expiration date is indicated on the on the indicated is date expiration The freeze. human CD146 antibodies (isotype: mouse IgG1). mouse (isotype: CD146human antibodies 68, 5142968, Fax Fax +49 2204 85197 2204 +49 Bergisch Gladbach, Germany Bergisch Gladbach, μm nylon filters. The cell suspension suspension cell The filters. nylon μm + cells from human human from cells

°C. Do not Do °C.

1.3 1.2 1.5 1.4 The CD146 MicroBead Kit was developed for the isolation of isolation for the developed was Kit CD146The MicroBead CD146 labeled magnetically The T lymphocytes.¹ T human CD146 Kit MicroBead

● ● ● ● ● ● CD146 possesses a limited tissue expression pericytes distribution, distribution, pericytes expression tissue alimited CD146 possesses and Mel-CAM, MUC18, as MCAM, known CD146 also antigen, CD146 Separator. of a MACS field magnetic the in placed is which Column, CD146 expression in is also directly associated with with associated directly also is melanoma CD146 in expression aCa as CD146 functions MicroBeads. Then, the cell suspension is loaded onto a MACS® onto aMACS® loaded is suspension cell the Then, MicroBeads. which is involved in heterophilic cell-cell interactions⁷, and may may and interactions⁷, cell-cell heterophilic in involved is which First, the CD146 the First, dendritic cells, melanoma cells, and a sub-population of activated of activated asub-population and cells, melanoma cells, dendritic of CD146depleted column. The unlabeled cells run through; this cell fraction is thus thus is fraction cell this through; run cells unlabeled The column. contain cells with clonogenic fibroblastic colony (CFU-F) activity colony (CFU-F) activity fibroblastic clonogenic with cells contain Blocking Reagent. The CD146 MicroBeads recognize the human human the recognize CD146 The MicroBeads Reagent. Blocking magnetic field, the magnetically retained CD146 retained magnetically the field, magnetic mesenchymal stromal cells (MSCs). cells stromal mesenchymal S-Endo-1. CD146 is a transmembrane glycoprotein belonging to to belonging glycoprotein S-Endo-1. CD146 atransmembrane is as the positively selected cell fraction. fraction. cell selected positively the as and multilineage differentiation potential, which are termed termed are which potential, differentiation multilineage and Sample preparation from lipoaspirate from preparation Sample the immunoglobulin superfamily and contains five extracellular extracellular five contains and superfamily immunoglobulin the tumor growth and metastasis.⁷ and growth tumor including endothelial cells, smooth muscle cells, follicular follicular cells, muscle smooth cells, endothelial including domains.¹ (Ig)-like immunoglobulin lipoaspirate⁵, dental pulp, or endometrial tissue⁶ are described to to described are tissue⁶ or endometrial pulp, dental lipoaspirate⁵, be involved in the extravasion and/or homing of activated T cells³. Tcells³. of activated homing and/or extravasion the in involved be page 1/5

▲ or HUVECs, as such cells, endothelial of human Isolation of CD146 Enrichment CD146 of Isolation Enzyme stop medium: Dulbecco’s Modified Eagles Medium Medium Eagles Modified Dulbecco’s medium: stop Enzyme Proved 4G NB (e.g. solution Collagenase digestion Collagenase (PBS). saline phosphate-buffered Sterile Background information Background Separation MACS® of the Principle Reagent and instrument requirements instrument and Reagent Applications (DMEM, # (DMEM, Grade, Serva # Serva Grade, www.miltenyibiotec.com/protocols. cells (MSCs) from lipoaspirate. (MSCs) from cells °C in a water bath. awater at 37 in °C enzyme Resolve units). units, please refer to the appropriate supplement in section General protocols at at protocols General section in supplement appropriate the to refer please units, pericytes from human endothelial tissue. endothelial human from pericytes

+ Note: cells. The consists of CD146 MicroBeads and FcR and of CD146 consists MicroBeads kit The cells. For information on the conversion of other catalytic units to Wünsch Wünsch to units catalytic other of conversion the on information For 130-091-437) containing 20% fetal bovine serum (FBS). serum bovine fetal 20% containing 130-091-437) , ³ CD146 ³ + cells are magnetically labeled with CD146 with labeled magnetically are cells + cells. After removing the column from the the from column the removing After cells. 17465.02): 0.3 U/mL in sterile PBS (Wünsch (Wünsch PBS 17465.02): U/mL sterile 0.3 in + cells from human tissues. human from cells + 2+ cells isolated from umbilical cord⁴, umbilical from isolated cells + cells, such as mesenchymal stromal stromal mesenchymal as such cells, -independent molecule -independent adhesion molecule cell , ⁸ + cells are retained within the the within retained are cells , ² Order no. 130-093-596 + cells can be eluted eluted be can cells 140-002-274.04 ▲ ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

are for research useonlyandnotfor diagnostic ortherapeutic use. MSC/ADSC cultivation CD146 using MicroBeads Separation MACS® Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products

XS LS selection Positive Column 10⁸ 10⁹ APC (#APC mM EDTA, pH 7.3. Always dilute freshly before use. before freshly pH EDTA, 7.3. dilute 1 mM Always VarioMACS™ or SuperMACS™ Separators. For details see the respective MACS MACS respective the see details For Separators. SuperMACS™ or VarioMACS™ NH Expansion Medium (# Medium Expansion NH (# Kit Removal Cell Dead (Optional) (# Solution Iodide Propidium (Optional) CD146, CD31, Fluorochrome-conjugated or (Optional) clumps. remove to cell (# Filters 130-041-407) Pre-Separation ▲ CD146 Separators: MACS and Columns MACS ▲ phosphate-buffered containing asolution Prepare Buffer: 75 (10×): buffer KHCO₃, mM 100 lysis 1.55 MNH₄Cl, Erythrocyte 37 to °C. pre-warmed Water bath, control. temperature with shaker Orbital # 352340). (e.g. BD Biosciences strainer cell μm 40 # 352360). (e.g. BD Biosciences strainer cell μm 100 # 352070). (e.g. BD Biosciences tubes conical mL 50 # 430518). (e.g. Corning bottles 1 L storage 500 (e.g. Corning # 431123).(e.g. Corning (# 222). Keep buffer cold (2−8 cold 222). buffer Keep 2 7-AAD for flow cytometric exclusion of dead cells. of dead exclusion cytometric 7-AAD for flow composition of sample the column capacity may be decreased. be may capacity column the sample of composition dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced replaced be (CPD). can BSA dextrose phosphate (ACD-A) citrate or formula-A dextrose CD34 antibody for flow cytometric analysis, e.g., CD146- e.g., analysis, cytometric for flow antibody CD34 Manual separation is recommended. recommended. is separation Manual www.miltenyibiotec.com. depletion of cells. dead cell culture flasks (e.g. BD Biosciences # 353109). (e.g. BD Biosciences flasks culture cell CD146 of selected expansion saline (PBS), pH saline 091-376) 1:20 with autoMACS® Rinsing Solution (# Solution 091-376) Rinsing autoMACS® with 1:20 information about other fluorochrome conjugates see see conjugates fluorochrome other about information Buffers or media containing Ca containing media or Buffers be enriched by using LS or XS Columns (positive selection). selection). (positive Columns or XS LS by using enriched be column. the block could bubbles Separator data sheet. data Separator by other such as human serum albumin, human serum, or fetal bovine serum. serum. bovine fetal or serum, human albumin, serum human as such proteins other by

mM EDTA by diluting MACS® BSA Stock Solution (# Solution Stock BSA MACS® EDTA by diluting mM Note: Note: Note: 130-092-652), or CD34-FITC (# or CD34-FITC 130-092-652), cm² cell culture flasks (e.g. BD Biosciences # (e.g. BD Biosciences flasks culture cell cm² mL Conical Bottom Centrifuge Tubes with screw caps caps screw Tubes with Centrifuge Bottom Conical mL

cells labeled of EDTA can be replaced by other supplements such as anticoagulant citrate citrate anticoagulant as such supplements other by replaced be can EDTA The capacities of the columns represent guidelines. Depending on the the on Depending guidelines. represent columns the of capacities The number Max. Column adapters are required to insert certain columns into the the into columns certain insert to required are adapters Column 130-092-849), CD31-PE130-092-849), (#

7.2, 0.5% bovine serum albumin (BSA), and albumin serum 7.2, bovine 0.5% 2+ Max. number Max. of total cells total of or Mg or 2 2 ×10¹⁰ ×10⁹ °C). Degas buffer before use, as air air as use, before buffer °C). Degas

+ 2+ 130-091-680) for the optimiz for the 130-091-680)

MSCs in culture. in MSCs are not recommended for use. for recommended not are

130-092-653), CD31-APC130-092-653), SuperMACS SuperMACS VarioMACS, QuadroMACS, MidiMACS, Separator 130-081-001). For more 130-090-101) for the 130-093-233 353136) cm² or 25 + cells can can cells 130-091- ) or 130-

ed ed

▲ ▲ ▲ ▲ ▲ 1. 4. 2. 3.

2.1 2. ● ● 6. 5. www.miltenyibiotec.com/protocols. °C (max. 24 hours) use.⁹ 24 before (max. °C at 2–8 or stored damage. cell unwanted to lead can at available is cord” umbilical from (HUVECs) cells endothelial for a sufficient yield of CD146 yield for asufficient stromal vascular fraction (SVF). fraction vascular stromal tumescent liposuction. Other methods, for example ultrasound, ultrasound, for example methods, Other liposuction. tumescent including the use of sterile reagents and media. and reagents of sterile use the including lipoaspirate will result in 1×10⁷ to 1×10⁸ mononuclear cells in the 1×10⁷ the in in result 1×10⁸ to cells mononuclear will lipoaspirate page 2/5 page

Lipoaspirate should be stored at room temperature (max. 4hours) (max. temperature at room stored be should Lipoaspirate A minimum starting volume of 250 mL of lipoaspirate is required required is of lipoaspirate mL of 250 volume starting A minimum All steps should be performed under sterile working conditions, conditions, working sterile under performed be should steps All For optimal results, only use aspirate that has been obtained by obtained been has that aspirate use only results, For optimal A special protocol for “Isolation of human umbilical vein vein umbilical of human for “Isolation protocol A special 1 (O Figure 1: Figure Protocol ▲ Apply to a fresh Conical Bottom Centrifuge Tube and dilute Tube dilute and Centrifuge Bottom Conical afresh Apply to for 10 at 430×g Centrifuge Dilute aspirated lipid fraction with an equal volume of the of the volume equal an with fraction lipid aspirated Dilute 3twice. 2and steps Repeat (Optional) NH Differentiation Media, e.g., NH AdipoDiff AdipoDiff NH e.g., Media, Differentiation NH (Optional) Incubate mixture at 37 mixture Incubate of and PBS volume equal an with sample lipoaspirate Dilute Preparation of the stromal vascular fraction (SVF) from from (SVF) fraction vascular stromal of the Preparation during incubation on the orbital shaker. orbital the on incubation during Medium (# Medium MSCs. with an equal volume of PBS. volume equal an with divide evenly between the Conical Bottom Centrifuge Tubes. Centrifuge Bottom Conical the between evenly divide optimized and reproducible quantification of selecte quantification reproducible and optimized of 250 Arotation shaker. orbital a to mixture the transfer and solution digestion collagenase phase lipid cell–containing target remove the centrifugation, from the top (see figure 1). (see top figure the from 091-679), or NH OsteoDiff Medium (# 130-091-678). Medium 091-679), OsteoDiff or NH thorough mixing and optimal digestion of the cell aggregates. cell of the digestion optimal and mixing thorough Note the upper (yellow) CD146 (yellow) upper the Note bottle. human lipoaspirate human be aspirated and washed a further two times. two afurther washed and aspirated be 500 more than not transfer Do bottle. L storage

Note: ptional) NH CFU-F Medium (# Medium CFU-F NH ptional)

Bottles should only be half filled in order to facilitate an optimal mixing mixing optimal an facilitate to order in filled half be only should Bottles Lipoaspirate preparation after the addition of PBS and centrifugation. centrifugation. and PBS of addition the after preparation Lipoaspirate 130-091-677), NH ChondroDiff Medium (# Medium ChondroDiff NH 130-091-677), + for 30 °C cell-containing lipid phase at the top which is to to is which top the at phase lipid cell-containing + cells. Starting with 250 mL of mL 250 with Starting cells. minutes without brakes. After After brakes. without minutes rpm should be used to ensure ensure to used be should rpm minutes on a pre-warmed on apre-warmed minutes 130-091-676) for the 130-091-676) for the Order no. 130-093-596 no. Order mL per 1 per mL d CD146 130- L +

140-002-274.04 ▲ ▲ ▲ ▲ 1. same the use 10⁷ cells, than fewer with working When cells. 10⁷ total 14. 13. 12. 11. 10. 4. 3. ▲ ▲ 2. 7. 7. 9. 6. 5. volumes and total volumes). total and volumes numbers, cell higher with working When indicated. as volumes are for research useonlyandnotfor diagnostic ortherapeutic use. which may clog the column. Wet filter with buffer before use. before buffer with Wet filter column. the clog may which on ice may require increased incubation times. Higher temperatures temperatures Higher times. incubation increased require may on ice labeling. cell 8. 8. mesh (Pre-Separation Filters, # Filters, (Pre-Separation mesh reagent indicated of all volume the twice use for 2×10⁷ cells, total suspension before magnetic separation. Pass cells through 30 µm nylon nylon µm 30 through cells Pass separation. magnetic before suspension (e.g. accordingly volumes total and volumes reagent up all scale and/or longer incubation times may lead to non-specific cell cell non-specific to lead may times incubation longer and/or prevent capping of antibodies on the cell surface and non-specific non-specific and surface cell on the of antibodies capping prevent labeling. Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products Work fast, keep cells cold, and use pre-cooled solutions. This will will This solutions. pre-cooled use and cold, cells keep Work fast,

2–8 is temperature incubation recommended The For optimal performance it is important to obtain asingle obtain to important it is performance For optimal Volumes for magnetic labeling given below are for up to are below given labeling Volumes for magnetic Aspirate and discard the supernatant. the discard and Aspirate Resuspend pellet (the stromal vascular fraction, SVF) in 10 in SVF) fraction, vascular (the stromal pellet Resuspend Mix well and incubate for 15 minutes in the refrigerator refrigerator the in for 15 minutes incubate and well Mix Conical fresh into preparation cell digested Redistribute Resuspend cell pellet in 60 µL of buffer per 10⁷ cells. 10⁷ per cells. of buffer µL 60 in pellet cell Resuspend Aspirate for 10 minutes. at 300×g suspension cell Centrifuge of 1× 1mL by adding cells blood Lyse red (Optional) Aspirate for 10 minutes. at 300×g suspension cell Centrifuge Add 20 µL of CD146 MicroBeads per 10⁷ total cells. 10⁷ per total of CD146 µL Add 20 MicroBeads tube. conical a15 into mL 10⁷Transfer cells to proceed CD146 to expression, according For isolation number. cell Determine a40 through suspension cell Pass for 10 at 600×g Centrifuge a100 through suspension cell Pass 30 After Add 20 µL of FcR Blocking Reagent per 10⁷ total cells. 10⁷ per total Reagent of FcR Blocking µL Add 20 (2–8 no erythrocytes are observed in the sample, proceed directly to step 5below. step to directly proceed sample, the in observed are erythrocytes no of PBS. of erythrocyte lysis buffer and incubating cells for 10 minutes at for 10 minutes cells incubating and buffer lysis erythrocyte Bottom Centrifuge Tubes and centrifuge at 600×g for 10 at 600×g Tubes centrifuge and Centrifuge Bottom room temperature. temperature. room significant erythrocyte contamination after preparation of SVF. If very few or or few very If SVF. of preparation after contamination erythrocyte significant pellet. medium to each bottle. each to medium magnetic labeling (2.2). labeling magnetic tubes. conical mL 50 fresh in filtrate supernatant completely. supernatant completely. supernatant of buffer. PEB 5 mL in pellet resuspend and supernatant mL conical tubes. tubes. conical mL 50 in filtrate the Note:

Note: °C). Erythrocyte lysis is only necessary when the sample contains contains sample the when necessary only is lysis Erythrocyte

More buffer may be applied if necessary, depending on the size of the the of size the on depending necessary, if applied be may buffer More 2.2 minutes, add an equal volume of the enzyme stop stop enzyme of the volume equal an add minutes, Magnetic labeling Magnetic 130-041-407) to remove cell clumps clumps remove to cell 130-041-407) minutes. Aspirate and discard discard and Aspirate minutes.

μm cell strainer and collect collect and strainer cell μm μm cell strainer and collect collect and strainer cell μm °C. Working Working °C. minutes. minutes. ‑

cell cell mL mL

▲ ▲ 1. 12. 11. 10. 4. 3.

2. 7. 9. 6. 5. For details see table in section 1.5. section in table see For details For instructions on the column assembly and the separation refer to to refer separation the and assembly column on the For instructions Magnetic separation with LS Columns LS with separation Magnetic Magnetic separation with XS Columns Columns XS with separation Magnetic according to the number of total cells and the number of CD146 number the and cells of total number the to according to the next step. next the to the XS Column data sheet. sheet. data Column XS the page 3/5 page

Always wait until the column reservoir is empty before proceeding proceeding before empty is reservoir column the until wait Always Separator MACS and Column MACS appropriate an Choose

Wash cells by adding 1 mL of buffer per 10⁷ cells and centrifuge centrifuge and 10⁷ per cells of buffer 1mL by adding Wash cells (Optional) Add staining antibody, e.g., 10 µL of 10 e.g., µL antibody, Add staining (Optional) ▲ ▲ Wash column with 3×3 with Wash column aMACS use Always column. onto the suspension Apply cell 3 with by rinsing column Prepare Remove column from the separator and place it on a suitable it on asuitable place and separator the from column Remove Pipette 5 Pipette MACS of asuitable field magnetic the in column Place (2.3). separation magnetic to Proceed 500 in pellet cell Resuspend (Optional) To increase the purity of CD146 purity the To(Optional) increase (# 130-092-849), and incubate for 5 minutes in the dark in the the in dark the in for 5minutes incubate and (# 130-092-849), cells (ADSCs) see special protocol “Isolation of CD271 (LNGFR CD271 of “Isolation protocol special see (ADSCs) cells buffer. of instead Medium Expansion NH of volume appropriate an using column Pre-Separation Filter. Collect unlabeled cells that pass through. pass that cells unlabeled Filter. Collect Pre-Separation collection tube. refrigerator (2–8 refrigerator using a new column. anew using magnetic separation procedure as described in steps 1 to 6by 1to steps in described as procedure separation magnetic the Repeat Column. LS over asecond enriched be can fraction reservoir is empty. is reservoir sheet. data Column MACS respective the see For details Separator. from human lipoaspirate” at www.miltenyibiotec. at lipoaspirate” human from at 300×g for 10 minutes. Aspirate supernatant completely. supernatant Aspirate for 10 minutes. at 300×g adding buffer three times. three buffer adding For further information regarding the cultivation of MSCs or adipose-derived stem stem adipose-derived or MSCs of cultivation the regarding information further For this is the unlabeled cell fraction. Perform washing steps by steps washing Perform fraction. cell unlabeled the is this the magnetically labeled cells by firmly pushing the plunger plunger the pushing by firmly cells labeled magnetically the into the column. the into

Note: Note: 2.3

Perform washing steps by adding buffer aliquots only when the column column the when only aliquots buffer adding by steps washing Perform For MSC/ADSC cultivation, it is recommended to flush cells from the the from cells flush to recommended is it cultivation, MSC/ADSC For

mL of buffer onto the column. Immediately flush out flush Immediately column. onto the of buffer mL Magnetic separation Magnetic °C).

mL of buffer. of buffer. mL

µL of buffer per 10⁷ per cells. of buffer µL

mL of buffer. mL com/protocols. Collect total effluent; effluent; total Collect + cells, the eluted eluted the cells, Order no. 130-093-596 no. Order CD146-AP + ) MSCs/ADSCs ) MSCs/ADSCs + cells. C

140-002-274.04 3. CD34-FITC (# 130-081-001). Cell debris and dead cells are excluded excluded are cells dead and (# debris 130-081-001).CD34-FITC Cell CD146 are for research useonlyandnotfor diagnostic ortherapeutic use. MicroBead Kit, an LS Column, and a MidiMACS™ Separator. Cells Cells Separator. a MidiMACS™ and Column, LS an Kit, MicroBead fluorescence. iodide propidium and signals on scatter based analysis the from are fluorescently stained with CD146-APC (# with stained fluorescently are Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products Example of a separation of aseparation Example using the CD146 the using Kit MicroBead + cells were isolated from lipoaspirate using the CD146 the using lipoaspirate from were isolated cells

CD34-FITC CD34-FITC CD34-FITC CD146 CD146 Before separation Before CD146-APC CD146-APC CD146-APC + – cell fraction cell cell fraction cell

130-092-851) and and 130-092-851)

References4. 1. Warnings 4. deposits in plumbing where explosive conditions may develop. may conditions explosive where plumbing in deposits Miltenyi Biotec provides technical support worldwide. Visit worldwide. support technical provides Biotec Miltenyi www.miltenyibiotec.com/local to find your nearest Miltenyi Biotec Biotec Miltenyi nearest your find to www.miltenyibiotec.com/local 2. Refer to to Refer contact. 7. 3. 9. 6. running water before discarding. These precautions are recommended to avoid avoid to recommended are precautions These discarding. before water running 5. Reagents contain sodium azide. Under acidic conditions sodium azide yields yields azide sodium conditions acidic Under azide. sodium contain Reagents 8. hydrazoic acid, which is extremely toxic. Azide compounds should be diluted with with diluted be should compounds Azide toxic. extremely is which acid, hydrazoic page 4/5 page Yan, X. Yan, X. Baksh, D. Baksh, Satyamoorthy, K. K. Satyamoorthy, Pickl, W. F. Pickl, Matsumoto, D. Matsumoto, Schwab and Gargett (2005) Prospective isolation of human endometrial endometrial human of isolation Prospective (2005) Gargett and Schwab N. Bardin, Xie, S. S. Xie, Zannettino, A. C. C. A. Zannettino, communication. Oncogene 20: 4676–4684. 20: Oncogene communication. angiogenesis and tumor growth. Blood 102: 184–191. 102: Blood growth. tumor and angiogenesis on liposuction aspirates. Plast. Reconstr. Surg. 120(6): 1510–1517. 120(6): Surg. Reconstr. Plast. aspirates. liposuction on 214: 413–421. 214: mesenchymal stem cells using CD146 and platelet-derived growth factor factor growth platelet-derived CD146 and using cells stem mesenchymal stem cells exhibit a perivascular phenotype phenotype aperivascular exhibit cells stem receptors. Reprod. Fert. Devel. 17: 111–111. Devel. Fert. Reprod. receptors. 1384–1392. 25: Cells Stem Marrow. Bone and Cord Umbilical Leads to Increased Tumor Growth and Metastasis. Cancer Res. 57: 2295–2303. Res. Cancer Metastasis. and Tumor Growth Increased to Leads junction involved in the control of cell-cell cohesion. Blood 98: 3677–3684. 98: Blood cohesion. cell-cell of control the in involved junction Differentiation Potential of Human Mesenchymal Stem Cells Derived from from Derived Cells Stem Mesenchymal Human of Potential Differentiation inhibit melanoma growth and invasion through loss of gap junctional junctional gap of loss through invasion and growth melanoma inhibit human T lymphocytes. J. Immunol. 158: 2107–2115. 158: Immunol. J. Tlymphocytes. human www.miltenyibiotec.com et al. al. et et al. et et al. et et al. al. et (1997) Expression of MCAM/MUC18 by Human Melanoma Cells Cells Melanoma Human by MCAM/MUC18 of (1997) Expression (2003) A novel anti-CD146 monoclonal antibody, AA98, inhibits inhibits AA98, antibody, monoclonal anti-CD146 Anovel (2003) et al. et (2001) Identification of CD146 as a component of the endothelial endothelial the of a component CD146 of as (2001) Identification et al. et et al. et (1997) MUC18/MCAM (CD146), an activation antigen of of antigen (CD146), activation an (1997) MUC18/MCAM et al. et (2007) Comparison of Proliferative and Multilineage Multilineage and Proliferative of Comparison (2007) (2007) Influences of preservation at various temperatures temperatures various at preservation of Influences (2007) (2001) Mel-CAM-specific genetic suppressor elements elements suppressor genetic (2001) Mel-CAM-specific (2008) Multipotential human adipose-derived stromal stromal adipose-derived human Multipotential (2008) for all data sheets and protocols. protocols. and sheets data for all in vitro in and and in vivo in Order no. 130-093-596 no. Order . J. Cell Physiol. Physiol. Cell . J.

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