CD34 Microbead Kit Ultrapure Contains Microbeads Microbeads Contains Ultrapure Kit Microbead CD34 the 1.2 1.1 1

140-004-115.03 1. The CD34 MicroBead Kit UltraPure contains MicroBeads MicroBeads contains UltraPure Kit MicroBead CD34 The 1.2 1.1 1. Description

Contents 2. Capacity Components 3. CD34-expressing cells from peripheral blood, cord blood, bone bone blood, cord blood, peripheral from cells CD34-expressing stem hematopoietic of human marker awell-established is CD34 MACS® Column which is placed in the magnetic field of a MACS of a field magnetic the in placed is which Column MACS® onto a is loaded suspension cell the Then, UltraPure. MicroBeads First, the CD34 the First, directly conjugated to CD34 antibodies for magnetic labeling of labeling for magnetic antibodies CD34 to conjugated directly endothelial progenitor cells, and mature endothelial cells. cells. endothelial mature and cells, progenitor endothelial Product format marrow, or apheresis harvest. Hematopoietic progenitors present present progenitors Hematopoietic harvest. or apheresis marrow, CD34 retained magnetically the field, magnetic macsde Separator. The magnetically labeled CD34 labeled magnetically The Separator. www.miltenyibiotec.com www.miltenyibiotec.com and progenitor cells and additionally expressed on hemangioblasts, on hemangioblasts, expressed additionally and cells progenitor and fraction. cell selected positively the as Storage thus depleted of CD34 depleted thus is fraction cell this through; run cells unlabeled The column. the Phone Phone Friedrich-Ebert-Straße Friedrich-Ebert-Straße Miltenyi Biotec B.V. KG &Co. Biotec Miltenyi Protocol Description Example of a separation using the CD34 MicroBead Kit - Kit MicroBead CD34 the using of aseparation Example Background information Background Separation MACS® of the Principle

2.4 2.3 2.2 2.1 1.4 Applications 1.3 1.2 1.1 UltraPure +49 2204 8306-0, 2204 +49 @

miltenyi.com

cell purity cell (Optional) Evaluation of hematopoietic progenitor progenitor of hematopoietic Evaluation (Optional) separation Magnetic labeling Magnetic preparation Sample requirements instrument and Reagent information Background Separation MACS® of the Principle

+ cells are magnetically labeled with CD34 CD34 with labeled magnetically are cells For 2×10⁹ total cells, up to 20 separations. up20 to For cells, 2×10⁹ total Store protected from light at 2−8 light from Store protected Human IgG. Human 2 mL FcR Blocking Reagent, human: Reagent, Blocking FcR 2 mL 2 mL CD34 MicroBeads UltraPure, UltraPure, MicroBeads CD34 2 mL CD34 MicroBeads are supplied in buffer buffer in supplied are MicroBeads CD34 vial label. vial MicroBeads conjugated to monoclonal mouse mouse monoclonal to conjugated MicroBeads IgG1) containing stabilizer and 0.05% sodium azide. 0.05% sodium and stabilizer containing freeze. The expiration date is indicated on the on indicated is date expiration The freeze. anti- 68, 5142968, Fax Fax + cells. After removing the column from the from column the removing After cells. +49 2204 85197 2204 +49 human CD34 human . Bergisch Gladbach, Germany Bergisch Gladbach, antibodies (isotype: mouse mouse (isotype: antibodies + cells are retained within within retained are cells + cells can be eluted eluted be can cells °C. Do not Do °C. human :

The CD34 MicroBead Kit UltraPure is suited for all routine routine all for suited is UltraPure Kit MicroBead CD34 The 1.4 Applications 1.3 human UltraPure CD34 Kit MicroBead (MNCs) or 0.5–3% among bone marrow MNCs can be rapidly and and rapidly be can MNCs marrow bone among (MNCs) or 0.5–3% page 1/4 ● ● ● ● ● ● ● ● CD34 efficiently enriched. enriched. efficiently cells (PBMCs), mononuclear blood cord cells 0.1–0.5% among at a frequency of 0.05–0.2% among peripheral blood mononuclear mononuclear blood peripheral among of 0.05–0.2% at a frequency particular advantages with debris-rich sample material. sample debris-rich with advantages particular

▲ ▲ 1:20 with autoMACS® Rinsing Solution (# Solution Rinsing autoMACS® with 1:20 VarioMACS™ or SuperMACS™ II Separators. For details refer to the respective respective the to refer details For Separators. II SuperMACS™ or VarioMACS™ Isolation of CD34 of Isolation vitro In Is from especially cells, progenitor hematopoietic of Isolation CD34 of Isolation CD34 human expressing of cells or depletion selection Positive MACS Columns and MACS Separators: CD34 Separators: MACS and Columns MACS saline phosphate-buffered containing solution a Prepare Buffer: Reagent and instrument requirements instrument and Reagent (PBS), pH can be replaced by other proteins such as human serum albumin, human serum, serum, human albumin, serum human as such proteins other by replaced be can (CPD). BSA dextrose phosphate (ACD-A) citrate or formula-A dextrose citrate or fetal bovine serum (FBS). Buffers or media containing Ca containing media or Buffers (FBS). serum bovine fetal or EDTA by diluting MACS BSA Stock Solution (# Solution Stock BSA MACS EDTA by diluting Cells that strongly express the CD34 antigen can also be be also can antigen CD34 the express strongly that Cells depletion can also be performed by using the autoMACS Pro Pro autoMACS the by using performed be also can depletion or selection Positive Columns. or XS LS, MS, using depleted Separator. autoMACS or the selection). (positive Columns or XS LS, MS, by using enriched column. the block could recommended for use. for recommended antigen. MACS Separator data sheet. data Separator MACS iPS cell cultures. cell iPS low frequency samples. low frequency buffer cold (2−8 buffer autoMACS XS Column LS MS Positive selection Positive selection +

olation of endothelial progenitor cells (EPCs). cells progenitor endothelial of olation

cell isolations. In addition, its unique formulation provides provides formulation unique its addition, In isolations. cell

Note: Note: Note: Note: differentiation studies. differentiation EDTA can be replaced by other supplements such as anticoagulant anticoagulant as such supplements other by replaced be can EDTA Column adapters are required to insert certain columns into the the into columns certain insert to required are adapters Column 7.2, 0.5% bovine serum albumin (BSA), 2 and albumin serum 7.2, bovine 0.5% 10⁷ 10⁸ Max. number number Max. 10⁹ of labeledcells 2×10⁸ °C). Degas buffer before use, as air bubbles bubbles air as use, before buffer °C). Degas + + progenitor cells from differentiated ES and ES differentiated from cells progenitor cells from debris-rich samples. debris-rich from cells 2 ×10⁹ 2 ×10⁸ of total cells 2 ×10¹⁰ Max. number 4 ×10⁹ Order no.Order 130-100-453 autoMACS Pro,autoMACS autoMACS SuperMACS II SuperMACS VarioMACS, II SuperMACS VarioMACS, Separator MidiMACS, QuadroMACS, MiniMACS, OctoMACS, 130 ‑ 091 2+ + II cells can be be can cells or Mg or 130 ‑ 222). Keep ‑ 091 2+ are not not are ‑ mM mM 376) 140-004-115.03 ▲ ▲ ▲ 10⁸ total cells. When working with fewer than 10⁸ cells, use the same same the use 10⁸ cells, than fewer with working When cells. 10⁸ total 1. When working with tissues or lysed blood, prepare a single-cell asingle-cell prepare blood, or lysed tissues with working When coat, or buffy blood peripheral anticoagulated with working When 2. 2.1 2. ● ● ● ● ● volumes and total volumes). total and volumes numbers, cell higher with working When indicated. as volumes are for research useonlyandnotfor diagnostic ortherapeutic use. For details refer to the protocols section at www.miltenyibiotec.com/ section protocols the to refer For details density gradient centrifugation, for example, using Ficoll-Paque™. Ficoll-Paque™. using for example, centrifugation, gradient density cell labeling. labeling. cell material leukapheresis from of cells Preparation remove cell clumps which may clog the column. Moisten filter with with filter Moisten column. the clog may which clumps remove cell nylon mesh (Pre-Separation Filters, 30 µm # µm 30 Filters, (Pre-Separation mesh nylon for 2×10⁸ total cells, use twice the volume of all indicated reagent reagent indicated of all volume the twice use cells, for 2×10⁸ total 30 through cells Pass labeling. magnetic before suspension (e.g. accordingly volumes total and volumes reagent up all scale methods. standard using suspension prevent capping of antibodies on the cell surface and non-specific non-specific and surface cell on the of antibodies capping prevent protocols. by (PBMCs) isolated be should cells mononuclear blood peripheral Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products buffer before use. before buffer

Volumes for magnetic labeling given below are for up to are below given labeling Volumes for magnetic will This solutions. pre-cooled use and cold, cells keep Work fast, For optimal performance it is important to obtain asingle obtain to important it is performance For optimal ▲ (Optional) Pre-Separation Filters, 30 µm (# µm 30 Filters, Pre-Separation (Optional) (# Kit Removal Cell Dead (Optional) (# Solution Iodide Propidium (Optional) for flow antibodies Fluorochrome-conjugated (Optional) (# Cocktail Cell MC Stem CD34 (Optional) Protocol Wash cells once with buffer and resuspend in a final volume volume final in a and resuspend buffer with once Wash cells 30 through harvest apheresis Filter Sample preparation Sample 3-9-5) C4-IC (# (# CD45-FITC (# 130-090-854), 7-AAD for flow cytometric exclusion of dead cells. dead of exclusion cytometric 7-AAD for flow aspirate supernatant. Repeat step. washing CD133 (293C3)-PE (# (#CD34-PE flow cytometric analysis of separated cells. separated of analysis cytometric flow depletion of cells. dead of 300 clumps. cell (# CD34-FITC e.g., analysis, cytometric remove cell clumps. remove cell pellet in buffer and centrifuge at 200×g for for 10−15 200×g at centrifuge and buffer in pellet Separation Filters, 30 µm # µm 30 Filters, Separation antibodies. information about antibodies refer to www.miltenyibiotec.com/ to refer antibodies about information labeling.

130-080-201), or CD45-APC (# or130-080-201), CD45-APC Note: Note: µL of buffer for up to 10⁸ cells. Proceed to magnetic magnetic to Proceed cells. for to up of buffer 10⁸ µL To remove platelets after density gradient separation, resuspend cell cell resuspend separation, gradient density after To platelets remove 2.2 Magnetic labeling Magnetic 130-081-002), CD34-APC (# CD34-APC 130-081-002), 130-090-853), CD133130-090-853), (293C3)-PE 130-041-407), in order to remove to order in 130-041-407),

130-080-202), CD45-PE CD45-PE 130-080-202), 130-091-230). For more µm nylon mesh (Pre- mesh nylon µm 20 at minutes 130-090-101) for the 130-093-427) for for 130-093-427) 130-093-233) or 130-041-407) to to 130-041-407) 130-041-407) to to 130-041-407)

130-090-954), 130-090-954), 130-081-001), 130-081-001), °C. Carefully Carefully °C. ‑ cell cell µm µm

▲ ▲ ▲ 1. 1. 10. 4. 4. 3. 3. 2. 2. 7. 9. 6. 5. For details refer to the table in section 1.4. section in table the to refer For details page 2/4 page Magnetic separation with MS or LS Columns or LS MS with separation Magnetic 8. specific cell labeling. Working on ice may require increased increased may require on ice Working labeling. cell specific according to the number of total cells and the number of CD34 number the and cells of total number the to according temperatures and/or longer incubation times may lead to non- to lead may times incubation longer and/or temperatures proceeding to the next step. next the to proceeding incubation times. times. incubation

2–8 is temperature incubation recommended The Always wait until the column reservoir is empty before before empty is reservoir column the until wait Always Separator MACS and Column MACS appropriate an Choose

▲ Add 100 cells. for up 10⁸ to total Reagent of FcR Blocking Add µL 100 Apply cell suspension onto the column. Collect flow-through flow-through Collect column. onto the suspension Apply cell Wash cells by adding 5−10 by adding Wash cells Wash column with the appropriate amount of buffer. Collect Collect of buffer. amount appropriate the with Wash column

(Optional) Add fluorochrome-conjugated CD34 antibody antibody CD34 Add fluorochrome-conjugated (Optional) ▲ ▲ empty. is reservoir column Determine cell number. number. cell Determine Centrifuge cell suspension at 300×g for 10 at 300×g suspension cell Centrifuge AC136: # CD34-PE, for 30 incubate and well Mix Prepare column by rinsing with the appropriate amount of amount appropriate the with by rinsing column Prepare MACS suitable of a field magnetic the in column Place (2.3). separation magnetic to Proceed of buffer. µL 500 in up10⁸ to cells Resuspend for to up total of buffer 10⁸ µL 300 in pellet cell Resuspend

(2−8 CD45 antibody (e.g. antibody CD45 data sheet. completely. cells. cells. containing unlabeled cells. unlabeled containing recognizing another epitope than QBEND/10 (e.g. than clone epitope another recognizing unlabeled cells that pass through and combine with the flow- the with combine and through pass that cells unlabeled supernatant completely. supernatant Separator. For details refer to the respective MACS Column Column MACS respective the to refer For details Separator. and centrifuge at 300×g for 10 at 300×g centrifuge and µL of buffer. of µL 500 through from step 3. step from through incubate for 5 incubate buffer: Note: Note N ote: °C). °C). 2.3

For depletion with LD Columns, resuspend up to 1.25×10⁸ cells in in cells 1.25×10⁸ to up resuspend Columns, LD with depletion For Perfor For higher cell numbers, scale up buffer volume accordingly. accordingly. volume buffer up scale numbers, cell higher For µL of CD34 MicroBeads UltraPure for up to 10⁸ total for up10⁸ to total UltraPure MicroBeads of CD34 µL Magnetic separation Magnetic m washing steps by adding buffer aliquots only when the the when only aliquots buffer adding by steps m washing minutes in the dark in the refrigerator (2−8 refrigerator the in dark the in minutes µL 3×500 MS: 5 MS: 130-081-002) or fluorochrome-conjugated or fluorochrome-conjugated 130-081-002) µL 00

CD45-FITC, # CD45-FITC, mL of buffer for up to 10⁸ cells cells for to up of buffer 10⁸ mL minutes. Aspirate supernatant supernatant Aspirate minutes. minutes in the refrigerator refrigerator the in minutes LS: 3×3 mL LS: 3 mL LS: 130-080-202), and and 130-080-202), minutes. Aspirate Aspirate minutes. Order no. 130-100-453 no. Order °C. Higher Higher °C.

+ cells. °C).

140-004-115.03 ▲ ▲ ▲

7. 6. 5. are for research useonlyandnotfor diagnostic ortherapeutic use. For instructions on the column assembly and the separation refer refer separation the and assembly column on the For instructions cells. For details refer to the section describing the cell separation separation cell the describing section the to refer For details cells. labeled of magnetically frequency the and labeling, of magnetic Magnetic separation with the autoMACS® Pro Separator Separator Pro autoMACS® the with separation Magnetic Columns XS with separation Magnetic or the autoMACS® Separator autoMACS® the or use the autoMACS® Pro Separator or the autoMACS Separator. autoMACS or the Separator Pro autoMACS® the use autoMACS Separator should have a temperature of ≥10 °C. have atemperature should Separator autoMACS programs in the respective user manual. manual. user respective the in programs sheet. data Column XS the to Unless otherwise specificallyindicated,Miltenyi all Unless otherwise Biotec andservices products

Program choice depends on the isolation strategy, the strength strength the strategy, isolation on the depends choice Program the or Separator Pro the autoMACS for operating used Buffers on how to for instructions manual user respective the to Refer

1. 3. 2. 1. 3. 2. Magnetic separation with the autoMACS® Separator autoMACS® the with separation Magnetic Separator Pro autoMACS® the with separation Magnetic (Optional) To increase the purity of CD34 purity the To(Optional) increase Pipette the appropriate amount of buffer onto the column. column. the onto of buffer amount appropriate the Pipette Remove column from the separator and place it on a suitable it on asuitable place and separator the from column Remove

Repeat the magnetic separation procedure as described in in described as procedure separation magnetic the Repeat cells labeled magnetically the out flush Immediately collection tube. fraction can be enriched over a second MS or LS Column. Column. or LS MS over asecond enriched be can fraction steps 1 to 6 by using a new column. anew 6by 1to using steps column. the into the plunger pushing by firmly Positive selection of CD34 selection Positive Positive selection of CD34 selection Positive instrument. the prime and Prepare Collect positive fraction in row C of the tube rack. tube row Cof the in fraction positive Collect of CD34 selection Positive following one of the choose separation For astandard containing Apply tube instrument. the prime and Prepare Collect positive fraction from outlet port pos2. port outlet from fraction positive Collect of CD34 selection Positive following one of the choose separation For a standard containing Apply tube Posseld2. Place fractions. cell unlabeled and labeled the collecting Posseld2. C. Band rows in tubes collection Place fractions. cell unlabeled and labeled the collecting sample tube at the uptake port and the fraction collection collection fraction the and port uptake at the tube sample fraction the and rack tube row Aof the in tube sample programs: pos2. port neg1 and at port tubes programs: bone marrow or leukapheresis: Posseld. or leukapheresis: marrow bone Posseld. or leukapheresis: marrow bone 1 mL MS:

the sample and provide tubes for tubes provide and sample the for tubes provide and sample the + + cells from peripheral blood, blood, peripheral from cells cells from peripheral blood, blood, peripheral from cells + + LS: 5 mL LS: cells from cord blood: blood: cord from cells cells from cord blood: blood: cord from cells + cells, the eluted eluted the cells,

A comparison of the ungated forward scatter versus side scatter scatter side versus scatter forward ungated of the A comparison The purity of the isolated hematopoietic progenitor cells can be can cells progenitor hematopoietic isolated the of The purity 3. 3-8-0) CD34 # 130-080-202).

2.4 UltraPure (# UltraPure Use the antibodies in appropriate concentrations as recommended recommended as concentrations appropriate in antibodies Use the CD34-PE, clone: AC136, clone: # 130-081-0 CD34-PE, MiniMACS™ Separator. Cells were stained with CD34-PE (# CD34-PE with were stained Cells Separator. MiniMACS™ Miltenyi Biotec provides technical support worldwide. Visit worldwide. support technical provides Biotec Miltenyi (# Kit MicroBead washed and resuspended in buffer. in resuspended and washed www.miltenyibiotec.com/local to find your nearest Miltenyi Biotec Biotec Miltenyi nearest your find to www.miltenyibiotec.com/local UltraPure. Kit MicroBead CD34 the with For optimal discrimination of CD34 discrimination For optimal Isolation of CD34 of Isolation Refer to to Refer page 3/4 page contact. compared to lymphocytes. to compared (e.g. CD45 CD45 against antibody an with cells counterstain CD34 of Analysis microscopy. or fluorescence cytometry by flow evaluated Solution (#Solution be should cells staining, After fluorescent sufficient. be should staining using an antibody recognizing an epitope different from different epitope an recognizing antibody an using staining 081-002), CD45-FITC (#081-002), CD45-FITC propidium iodide-, lymphoid cells lymphoid iodide-, propidium a and Columns, MS two UltraPure, Kit MicroBead CD34 the profile of the same sample after separation with the CD34 the CD34 with separation after sample same the of profile that recognized by the CD34 monoclonal antibody QBEND/10 monoclonal CD34 by the (e.g. recognized that by the manufacturers. Typically, staining for 5 staining Typically, manufacturers. by the

Side scatter Example of a separation using using of aseparation Example (Optional) Evaluation of hematopoietic progenitor cell cell progenitor of hematopoietic Evaluation (Optional) CD45-FITC UltraPure CD34 Kit MicroBead purity 10 10 10 -1 1 0 ² ³ ¹ -1 www.miltenyibiotec.com + cells can be accomplished by direct immunofluorescent immunofluorescent by direct accomplished be can cells Kit MicroBead CD34 0 Before separation Before Fo 130-093-233). Cells were analyzed after gating on gating after were analyzed 130-093-233). Cells 1 130-100-453) demonstrates reduced debris carry-over carry-over debris reduced 130-100-453) demonstrates rw ard scatter CD34-PE 10 + ¹1 cells from a debris-rich PBMCs sample using using PBMCs sample adebris-rich from cells 130-046-702) or the CD34 MicroBead Kit Kit MicroBead CD34 or the 130-046-702) + 0² cells express CD45 at a lower level as as at alower level CD45 express cells

130-090-202) and Propidium Iodide Iodide Propidium and 130-090-202) 10 ³

for all data sheets and protocols. protocols. and sheets data for all

Side scatter CD45-FITC 02 + CD34 MicroBead Kit Ultra Pure Ultra Kit MicroBead CD34 10 10 10 cells from other leukocytes, leukocytes, other from cells ) -1 1 0 . ² ³ ¹ -1 0 the Fo After separation After 1 rw

ard scatter CD34-PE

minutes at 2 minutes 10 ¹1 Order no. 130-100-453 no. Order 0² 10 ‑ − FITC, FITC, ³ °C 8 °C 130-

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All All page 4/4 page Order no. 130-100-453 no. Order