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CD14 Isolation Kits

Contents

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

Other Equipment and Reagent Requirements

1.  Magnetic Separator
2.  Column
3.  Sterile serological and Pasteur pipettes or transfer pipettes
4.  30uM Filter (Partec, Catalog #04-0042-2316)
5.  Bench top centrifuge
6.  2 - 8° C refrigerator
7.  Deionized or distilled water


Applications

The COL-isoTM Human PBMC CD14+ Cells Isolation Kit is designed to isolate CD14+ human PBMC cells using positive selection. The resulting cell preparation is highly enriched for CD14+ cells. Purity of recovered CD14+ cells can be up to 97%-99% and will vary depending on the preparation.

Cell Selection Principle

  • Positive selection of CD14+cells is achieved by incubation with biotinylated anti-Human CD14 monoclonal antibody.
  • CD14monoclonal antibody bound cells are then magnetically tagged with COL-isoLTM -Streptavidin.

  • Magnetically tagged CD14+cells are then retained in themagnetic column. (These are the desired cells); unwanted/untagged cells run through.
  • Upon removal of column from magnetic field, CD14+ cells can then be eluted.

Cell Selection Capacity

Separator Max No. of CD14+ cells/column Max No. of cells/column
Column/EA *1x107 *1x108
*: The Max No. of cells will vary by ±40% depending on the preparation.
Components (for 109 cells, up to 100 tests).
  • Biotinylated anti-Human CD14 Antibody (Part C10101)-2mL
  • COL-isoTM- Streptavidin. (Part B10002) - 2mL proprietary formulation.

  • DRNase (proprietary formulation of DNase I and RNase)-1mL (Part DR10100)
Storage
Reagents except DRNase are stable for 6months from the date of receipt when stored in the dark at 2 - 8° C. DO NOT FREEZE. DRNase can be stored in -20° C .

Reagent Preparation

Selection Buffer: Phosphate buffered saline (PBS),pH 7.2, 0.5% bovine serum albumin (BSA) and 2 mM EDTA. Filter before use. Selection Bufferis stable for 6 months at 4?C and should be kept on ice or at 4?C throughout the selection process.

Cell Selection Procedure
  • Cell Preparation: 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°C 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 10μL formulated DRNase per 107 cells
  • Transfer desired amount of cellsuspension to the 50ml conicaltube.
  • Drop wise add 15mL pre-warmed (37?C) DMEM containing 10% FBS to the cells with constant swirling.
  • Centrifuge cell suspension at 300 x g at 4?C for 15 minutes.
  • Carefully remove all but approximately 100μL of the supernatant using a pipette.
  • Gently resuspend 107 cells with 80uL COLD Buffer.
  • Pre-wet a 30-50μm nylon cell strainer then pass the suspended cells through the strainer.
B. Preparing a cell suspension from fresh   PBMNC/mPBMNC/CB MNC.
  • Centrifuge cell suspension at 300 x g at 4?C for 15minutes.
  • Gently resuspend 107 cells with 80uL COLD Buffer.
  • Pre-wet a 30-50μm 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 times.

NOTE:For downstream applications that are sensitive to DRNase (eg. hematopoietic colony assays), wash cells once in the appropriate assay buffer (without DRNase) before continuing.

Technical Help and Suggestions

IV. FACS Analysis - Cell Staining

Cells can be stained by traditional methods or by following the instructions below.
  • Per 5x105cells, resuspend cell pellet in 80uL staining buffer (Part S10002; not included)
  • Add 20uL of the FITC-conjugated Human CD14 Detection Antibody (Part L10101, not included) to 106 cells.
  • Incubate in dark for 20 minutes at 2 - 8°C.
  • Dilute cell suspension in 1.5mL of cold staining buffer and centrifuge at 4°C at 300 x g for 5minutes.
  • Remove supernatant and resuspend pellet in 1.5mL cold staining buffer and centrifuge at 4°C at 300 x g for 5 minutes.
  • Remove supernatant and resuspend pellet to a final volume of 500uL staining bufferfor FACS analysis.

V. Cell Cryopreservation

2x freezing media preparation:

•  20% fetal bovine serum
•  20% DMSO
•  60% DMEM or RPMI media

Store freezing media at –20?C and thaw to 37?C or 4?C before use.

  • Add equal volume of 2x freezing medium to cell suspension or resuspend up to 106selected cells in 1mL of 1x freezing medium.
  • Store desired cells in cryovials.
  • Freeze cellsin a cryopreservation unit overnight at -80°C.
  • Transfer the frozen vial into liquid nitrogen tank the day after for long term storage.
II. Magnetic labelling of CD14+ cells n
  • Transfer desired amount PBMC cellsto an Eppendorf tube.
  • Add 20uL of biotinylated anti-human CD14 antibody (Part C10101) per 107cells.
  • Gently mix the cell-antibody suspension, avoiding formation of bubbles, and incubate at 2-8?C on a rotator for 15 minutes.
  • After incubation, wash cells by adding 1-2mL of buffer per 107 cells and centrifuge at 4°C at 300 x g for 10 minutes.
  • Carefully remove supernatant and resuspend 107 cells in 80uL of buffer.
  • Add 20 µL COL-isoTM-Streptavidin (Part B10002) per 107 cells.
  • Mix gently and incubate at 2 - 8°C on a rotator in a refrigerator for 15 minutes.
  • After incubation, wash cells by adding 1-2mL of buffer per 107cells and centrifuge at 4o C at 300 x g for 10minutes.
  • Completely remove supernatant and gently resuspend cell pellet up to 108 cells in 500uL of buffer.

III. Magnetic Separation

  • Place column in magnetic field. Prime column by rinsing 1x with 500uL of filtered Buffer.
  • Load up to 108cell suspension nbsp;onto each equilibrated column. (i.e. 2x108 cells nbsp;would require the use of 2 columns) Carefully save nbsp;effluent as Flow Through.
  • Wash column 3x with 500uL of cold buffer. Only apply 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 CD14+ cells with plunger. Label tube as Elution.
  • Centrifuge Flow Through and Elution at 8°C at 300 x g for 5 minutes.
  • Cells are now ready for further experimentation or FACS analysis.

Example Data

FACS analysis of PBMNC pre- and post- CD14+ cell selection.

A. Dot Plots overlay of pre-selected cells (red) and CD14+enriched (green) cells.

B. Histogram overlay of pre-selected cells (red) and CD14+ enriched (green) cells.