Quick-SepTM Goat Anti-Mouse (GAM) IgG Ferromagnetic Particles (FMP)

Package Insert 

Procedure for Purging Cells by Indirect Depletion

(Prelabel cells with desired mouse IgG monoclonal antibodies) 

Russell Biotech’s Quick-SepTM Ferromagnetic Cell Selection Technology is based on particles made of NICKEL. The technology is superior to current magnetic separation technologies because of three key properties of NICKEL: density, magnetic strength and particle surface.  For further information on the “Power of NickelSM” visit our website (www.russellbiotech.com ) .  

Product Number:  C00001 (1mL); C00002 (2mL)

Shelf Life:  One Year

NOTE: For research use only. Not for use in human diagnostic or therapeutic procedures.

IMPORTANT: DO NOT ALLOW PARTICLES TO COME IN CONTACT WITH A MAGNETIC FIELD UNTIL DIRECTED TO DO SO IN THE PROTOCOL.

A. Protocol for Indirect Depletion:

  1. Label cells (mononuclear cells or tissue culture cells) with desired mouse IgG monoclonal antibodies (mab(s)), rinse and resuspend cells (up to 2.5x107 total cells/ml) using standard procedures in your lab. Use cell buffer or PBS/0.1%BSA/pH 7.2 to resuspend cells.
  2. Rinse (section B) the required amount of GAM FMP (50ul/ml cell suspension with total cell concentration up to 2.5x107 cells/ml).* Vortex bottle well before removing particles. Following the rinsing step, resuspend GAM FMP to the original volume.
  3. Add GAM FMP (vortex well before removing particles) to cells (50ul/ml cell suspension).
  4. Immediately mix either by vortexing (sample volume</= 0.5ml) for1-2 minutes or end-over-end mixing (sample volume > 0.5ml) for 4 minutes (section C1)*.
  5. Immediately place in the appropriate magnetic separator (section C2) for two minutes.*
  6. Carefully remove the sample while still in the magnetic field to obtain the desired depleted sample.

*This amount of GAM FMP/ml is recommended as a starting point. The actual amount of particles/ml, mixing time and/or magnetic separation time should be varied to determine the best parameters for the cell type/ mab(s) used.

B.  Procedures for rinsing particles prior to addition to sample:

Particles should be rinsed prior to use in the indirect purging procedure. Quick-SepTM GAM FMP can be rinsed in three different ways because of the nature of the particles.  Step 1 or Step 2 is recommended. To save time, rinsing with magnetic separation (Step 3) is possible but the particles must be demagnetized prior to use (section C3). Rinsing buffer is PBS/0.1%BSA/pH 7.2. Note: Particles should not be stored in this buffer. Final resuspension buffer can be PBS/BSA buffer or cell buffer.

  1. Centrifugation: Centrifuge at 500rpm for 5 minutes. Following centrifugation, remove buffer, add new buffer and resuspend particles by vortexing or pipette up and down. Repeat two times. Resuspend to original volume using your working buffer (i.e. PBS/BSA or cell buffer).
  2. Gravity Settling: Allow particles to settle by gravity for 2-3 minutes. Following settling, remove buffer, add new buffer and resuspend particles by vortexing or pipette up and down. Repeat two times. Resuspend to original volume using your working buffer (i.e. PBS/BSA or cell buffer).
  3. Magnetic Separation: Place particles in magnetic separator for 4-5 seconds. Following magnetic separation, remove buffer, remove test tube from magnetic separator, add buffer and resuspend by vortexing or pipette up and down. Repeat two times. Resuspend to original volume using your working buffer (i.e. PBS/BSA or cell buffer). Demagnetize the final particle suspension (section C3). 

C.  Equipment required for optimal performance of Quick-SepTM FMP:

1. Mixer:  Due to the density difference between GAM FMP and cells (~8 fold), proper mixing is essential to ensure contact between the particles and the targeted cells. 

·         For reaction volumes < to 0.5ml mixing can be accomplished by vortexing at a low speed using a Vortex Genie 2 that has variable speed control. A Vortex Genie 2 with a timer is desirable but a timer is not essential.

·         For volumes > 0.5ml up to 50ml mixing can be accomplished by end-over-end mixing using an ATR Rotamix mixer (www.atrbiotech.com/benchtop/rotamix.htm) with variable speed. Recommended mixing speed is 15-30 rpm.

2. Magnetic Separation: 

·         Ideal magnets that can be used with Quick-SepTM FMP can be obtained from Dexter Magnetic Technologies (www.lifesep.com under products). Different magnets are available for sample volumes from < 0.5mL to 50mL

·         Magnets from suppliers of superparamagnetic particles will also work with Quick-SepTM Ferromagnetic Particles.

3. Demagnetizer: 

To ensure well dispersed particles, it is recommended, but not required unless particles inadvertently come in contact with a magnetic field prior to use, that the particles be demagnetized immediately prior to the addition to the sample, using a demagnetizer/degausser from Data-Link Associates (www.datalinksales.com ); product number PF-211.

Hold the demagnetizer at the bottom of the test tub containing the particles; (hold test tube in one hand; demagnetizer in the other hand; demagnetizer can touch test tube); turn unit on by holding “on button” in the on position; rotate demagnetizer in a clockwise or counterclockwise motion for 10-12 seconds; WHILE UNIT IS STILL ON (if unit is turned off before this step particles will be magnetized) slowly move the demagnetizer away from the test tube (about 3 feet; arms length); turn off demagnetizer.

 

For Technical Information/Questions contact Russell Biotech:

Phone (215) 589-612 or E-mail trussell@russellbiotech.com