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CD34 Isolation Kits

Contents

Other Equipment and Reagent Requirements
Applications
Cell Selection Principle
Column Capacity
Components
Storage
Reagent Preparationfrom
Cell Selection Procedure
Technical Help and Suggestions
Cell Staining Procedure
Cell Cryopreservation Procedure
Example Data

The COL-isoTM Human CD34+ Cells Isolation Kit is designed to isolate human CD34+ stem/progenitor cells from peripheral blood mononuclear cell (PBMNC), mobilized PBMC (mPBMNC) or cord blood mononuclear cells or (CBMNC) using positive selection. Purity of recovered CD34+ progenitor cells can be up to 98%.

Cell Selection Principle

  • Positive selection of CD34+ cells is achieved by incubation with biotinylated anti-Human CD34 monoclonal antibody.
  • CD34monoclonal antibody bound cells are then tagged with COL-isoTM -Streptavidin labeled magnetic nanoparticle beads.
  • Magnetically tagged CD34+ cells are then retained in the magnetic column. (These are the desired cells); unwanted/untagged cells run through.
  • Upon removal of column from magnetic field, CD34 + cells can then be eluted.

Cell Selection Capacity

Separator Max No. of labeled cells* Max No. of total cells**
Column/EA 5x106 1x108
 

Other Equipment and Reagent Requirements

  • Magnetic Separator
  • Column
  • Sterile serological and Pasteur pipettes or transfer pipettes
  • 30uM Filter (Partec, Catalog #04-0042-2316)
  • Bench top centrifuge or equivalent
  • Deionized or distilled water
  • Eppendorf Microcentrifuge 5415C

*.The purified number of CD34+ cells are usually lower than 5x106 when starting from 2x108 total cells

** To isolate CD34+ cell from one unit of umbilical cord blood/PBMC with 2x108 cells or above, multiple columns are needed

Components (for up to 10 tests with 2x108 cells per test)

  • Biotinylated anti-Human CD34 Antibody (Part C10134)- 1mL x 2 vials.
  • COL-isoTM- Streptavidin labeled nanoparticle magnetic beads (Part B10002) - 1mL x 2 vials proprietary formulation.
  • FcR Blocking Reagent ? 1mL x 2 vials
  • 10x MAG-isoTM Buffer - 50 mL, proprietary formulation.
  • DRNase (proprietary formulation of DNase I and RNase)? 400?L.
Storage

Reagents except DRNase are stable for 12 months from the date of receipt when stored in the dark at 2 - 8℃. DO NOT FREEZE. DRNase can be stored in -0℃ for long-term purpose.


Reagent Preparation

1x COL-isoTM Buffer: Prepare 50 mL of 1x COL-isoTM Buffer for each sample by mixing 5mL of 10X COL-isoTM Buffer with 45mL of sterile deionized or distilled water. The 1x COLisoTM Buffer is stable for 6 months at 4℃.

PROCEDURE:

  • Cell Preparation: Recommended sample size= 2x108 cells per selection using two columns. Cells and reagents should be kept cold using an ice bath or a refrigerator unless otherwise specified. Incubations must be carried out at 2 - 8℃ in a refrigerator and not in an ice bath to avoid excessively low temperatures that can slow the kinetics of the optimized reactions.

    A.Preparing a cell suspension from   frozen PBMNC/mPBMNC/CBMNC
    • To a 50 mL conical tube add 30?L formulated DRNase.
    • Transfer desired amount cell suspension to the 50mlconical tube.
    • Drop wise add 15mL pre-warmed (37℃) DMEMcontaining 10% FBS to the cells with constant swirling.
    • Centrifuge cell suspension at 300 x g at 4℃ for minutes.
    • Carefully remove all but approximately 100?L of the supernatant using a pipette.
    • Gently resuspend 108 cells with 300uL COLD Buffer.
    • Pre-wet a 30-50?m nylon cell strainer then pass the suspended cells through the strainer.

Cells must be resuspended in cold reaction buffer prior to the antibody selection procedure. Buffer has to be kept on ice at all time.

 

II. Magnetic Labeling

  • Determine Cell Number.
  • Add 100uL of FcR blocking reagent per 108 cells
  • Add 100?L of biotinylated anti-human CD34 antibody per 108 cells.
  • Gently mix the cell-antibody suspension, and incubate at 2-8℃ on a rotator for 15 minutes.
  • After incubation, wash cells with 5-10 mL of buffer and centrifuge at 4℃ at 300 x g for 10minutes.
  • Carefully remove supernatant and resuspend cells in 500uL of buffer.
  • Add 100 ?L COL-isoTM-Streptavidin beads per 108 cells.
  • Mix gently and incubate at 2 - 8℃ on a rotator for 15 minutes.
  • After incubation, wash with 5-10 mL of buffer and centrifuge at 4℃ at 300 x g for 10minutes.
  • Completely remove supernatant and gently resuspend cell pellet up to 108 cells/mL

III. Magnetic Separation

  • Equilibrate column by rinsing column 1x with 500uL of degassed buffer
  • Load up to 108 cell suspension onto equilibrated column(For 2x108 cells, 2 columns are needed.) Save effluent as Flow Through
  • Wash column 3x with 500uL of cold Buffer at a time. Only add new buffer when column reservoir is empty. Collect effluent into Flow Through from step 2
  • At the end of the washing step, remove column from magnetic field and place column on a collection tube.
  • Add 1mL of buffer onto column and immediately flush out the CD34+ cells with plunger. Label tube as E1.
  • Equilibrate a new column and repeat magnetic separation steps 1 through 5 with E1 fraction. Save and label second elution as E2. Save all effluent into Flow Through from step 2.

  • (Optional, repeat II and III with modification) To increase purity of CD34+ cells, a second incubation with 25uL of antibody and then 25uL beads is highly recommended prior to magnetic separation with a third column. Washing step following antibody incubation and beads incubation has to be carried out in microcentrifuge at 300x g for 10min. Cell pellet is then resuspended in 500uL prior to column separation.

  • 7. Cells are now ready for    further experimentation or    FACS staining.

IV. FACS Analysis - Cell Staining

Cells can be stained by traditional methods or by following the instructions below.
  • Resuspend 105 ~ 106 cells in 90uL staining buffer (Part S10002; not included)
  • Add 10uL of the PE-conjugated Human CD34 Detection Antibody (Part L10004, not included)to suspension.
  • Incubate for 10minutes at 2 - 8℃.
  • Dilute cell suspension in 1.5mL of cold staining buffer.
  • centrifuge at 4o C at 300 x g for 5 minutes.
  • Remove supernatant and resuspend cell pellet in 500uL staining buffer.
  • Repeat step 5 and 6.
  • Sample now ready for FACS analysis

V. Cell Cryopreservation

Freezing mediumpreparation:

  • 90% fetal bovine serum
  • 10% DMSO
Store freezing media at -20 ℃ and thaw to 37 ℃ or 4℃ before use.
  • In a cryovial, resuspend up to 106 selected cells in 1mL of freezing medium.
  • Freeze cells in a cryopreservation unitovernight at -80℃.
 

3. Transfer frozen vial into liquid nitrogen tank within 24-48 hrs for long term storage.

Example Data

Fig1. FACS analysis of CD34+ cell isolated from fresh mobilized PBMNC, purity 98% (dead cells are excluded)

Fig 2. FACS analysis of CD34+ cell isolated from frozen cord blood MNC, purity 90.9%