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BMP Products and Cell Sorting
I. General Product
Description
Biomagnetic Particles (BMP) are a series of solid supports useful in magnetic
separation. BMP are ~ 1 um magnetic particles consisting of an iron oxide core with a
silane coating. The particle surface is functionalized with amine or carboxyl groups for
the covalent attachment of proteins, glycoproteins and other ligands with retention of
biological activity. BMP products are also supplied covalently attached to a number of
secondary antibodies, such as goat anti-mouse and goat anti-rat as well as streptavidin,
biotin and other binding proteins. All BMP solid phases are superparamagnetic, i.e. they
respond well to magnetic fields but do not themselves become magnetized. The inability to
become permanently magnetized permits repeated magnetic extraction without magnetically
induced aggregation. Efficient and rapid removal of BMP particles from suspension is
achieved through the application of an external magnetic field.
II. Principle of Magnetic Cell Sorting
BMP are particularly suitable for cell isolations using either a direct or an indirect
procedure. In the direct procedure, monoclonal or polyclonal antibodies to cell
surface antigens are covalently attached to BMP before incubation with the cell
suspension. In the indirect procedure, the cells to be isolated are pretreated with
the appropriate monoclonal or polyclonal antibodies and are subsequently magnetically
immunoprecipitated with the appropriate magnetic second antibody. Variations of the indirect
method include pretreating the cells to be isolated with biotin-labeled antibodies and
magnetically separating them with BMP Streptavidin. The target cells magnetically labeled
by either the direct or indirect method are easily isolated by applying a
rare earth magnet directly against the side of the tube or tissue culture flask such that
the cells are magnetically separated perpendicular to gravity. Most cell separations
require a 5-10 minute magnetic separation.
III. Magnetic Cell Sorting Guidelines
No single magnetic cell sorting protocol is applicable for all cell
separations. The best way to start is by following the general guidelines offered here
while referring to published protocols (See References). Each lab should then optimize
conditions, i.e. antibody concentrations, incubations times, particle-to-cell ratios to
achieve the desired results.
A. Preparation of Cells
Lymphocytes may be partially purified using a LeucoPREPä
Tube, ficoll gradient, nylon wool, or similar method. However, it may be possible to sort
cells directly from whole blood and other sources. Since any particular cell source will
have its own unique requirements for purification, procedures must be optimized. Ideally,
cell concentrations should NOT exceed 5 million total cells per ml. The denser the
mixture of cells, the more likely there will be nonspecific binding, clumping and trapping
of cells. The investigator should work with the most dilute cellular suspension possible.
Sorts or one million cells per ml are most common. If cells clump during washes with
sterile media, it may be due to the release of DNA by necrotic cells. These clumps can be
easily broken up with the use of 0.1% DNase in the cell medium.
B. BMP Preparation
BMP products are not claimed to be sterile and are supplied in buffers containing azide.
Although not sterile, conjugated BMP products are all prepared by procedures which
essentially result in a low bio-burden, i.e. glutaraldehyde conjugations. To remove azide
and to prepare the magnetic particles for cell sorting, wash the magnetic particles 2-3
times in an appropriate sterile culture medium (or buffer) containing antibiotics such as
penicillin, streptomycin or gentamycin. Washes MUST be performed using a BMP
magnetic separation unit. Do not centrifuge during wash steps.
Media and buffers containing 5-10% protein are recommended to reduce possible
non-specific binding. Be advised that too much protein in the medium may inhibit the
binding of particles to cells. Proteins such as human serum albumin, bovine serum albumin,
fetal bovine serum, or milk solids may be used to lower non-specific binding.
C. Positive and Negative Selection Using BMP
BMP products may be used in both the positive and negative selection of
cells. Depending upon antigen availability and the size of the target cell population, positive
and negative cell sorting applications may require 20-80 magnetic particles per
cell based on the total cell population. Multiple sorts may also be performed for both positive
and negative selection. BMP products contain approximately 5 x 108
magnetic particles per mg. Most BMP products are supplied at 1 mg BMP/ml and 5 mg BMP/ml
concentrations. Since the particle-to-cell ratio is based upon the TOTAL cell
population, the following sample calculation applies for both positive and negative
selection.
Sample Calculation for Positive or Negative Selection
Consider a system in which there is 1 x 107 total cells and in which the
target cell population is 30%. In this example we will use a ratio of 50 particles per
total cell and the product BMP Goat anti-Mouse IgG (code # 610-119-019) which is supplied
as a 1 mg BMP/ml preparation:
1 x 107 total cells x 50 particles per total cell = 5 x 108
magnetic particles required.
Since 1 mg/ml BMP contain 5 x 108 particles per mg, which is equivalent to 5
x 108 magnetic particles per ml, the volume of washed BMP required is:
5 x 108 magnetic. particles req'd. = 1.0 ml of BMP Goat anti-Mouse IgG
5 x 108 magnetic particles per ml
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D. Incubation Guidelines for Positive and Negative
Selection
1. Incubations should be performed on ice or at 4° C to minimize patching, capping
and phagocytosis. Also, cell viability may be best preserved by keeping the cells on ice.
However, room temperature or even 37° C may be optimal for certain cell types. The ideal
incubation temperature for sorting may vary with the application. Therefore, the
investigator may wish to investigate other temperatures should low yield and/or cell
viability become concerns.
2. Magnetic particles should be incubated with the cells for 15 to 30 minutes at 4° C.
Long incubations are not recommended as magnetic particles may detach from the target
cells as a result of cell surface changes over time. During incubation, gentle swirling of
the reaction vessel at 10 minute intervals will keep the BMP in suspension. (Continuous
rotation is not recommended).
3. To reduce non-specific binding and prevent trapping, cell sorts should be performed
in total volumes, which include the cell volume plus the BMP volume, greater than or equal
to 1 ml. Additional media or buffer should be added to volumes less than 1 ml to bring the
volume to at least 1 ml.
E. Separation Guidelines for Positive and
Negative Selection
1. Magnetic separation must be performed perpendicular to gravity with the pellet
formed on the side of the flask or tube. This technique is used to keep the unselected
cells from contaminating the magnetic pellet due to gravity. The magnet used should have a
strength greater than 20 megagauss Orstead. Laboratory Supermagnets™ for use with BMP
(code # BMP-00-001 thorough BMP-00-007) have been designed specifically for magnetically
separating cells and are recommended.
2. Separation times of 5-10 minutes are generally sufficient for complete separations.
A clear supernatant indicates that the separation is complete.
3. Once separation is complete, the supernatant should be removed without disturbing
the magnetic pellet. For this reason, vacuum aspiration is not recommended.
F. General Procedure for the Indirect Method for
Isolating Lymphocytes
This procedure is for negative or positive cell selection is best carried out in
sterile tissue culture tubes, or flasks. The following is an example of a two step
protocol which uses BMP Goat anti-Mouse IgG (code # 610-119-019). The target population is
assumed to be 20% of the total. All steps are done at 4° C. The example uses a
particle to total cell ratio of 50:1 for BMP Goat anti-Mouse IgG which is supplied at a
concentration of 1 mg/ml.
1. Approximately 10 million total cells are placed in a tube in 10 ml of RPMI with
5% fetal bovine serum and antibiotics.
2. Wash 1 ml of BMP Goat anti-Mouse IgG three times in 1 ml of sterile medium
containing antibiotics. Use a magnet to pull the magnetic particles to the side of the
tube and shake vigorously to resuspend the magnetic particles during washing. Resuspend in
1 ml of sterile medium.
3. Depending upon the source of the antibody and the manufacturer’s
recommendations, 5-20 µg of monoclonal antibody per one million target cells is typically
needed. Add the appropriate amount of monoclonal antibody to the washed 1 ml of BMP Goat
anti-Mouse IgG from step 2 and mix. Incubate at 4° C for 20 minutes.
4. Magnetically separate the BMP Goat anti-Mouse IgG/antibody complex and wash three
times with 1 ml of sterile medium. Resuspend in 1 ml of sterile medium.
5. Add the 1 ml of washed BMP Goat anti-Mouse IgG/antibody complex to the 10 ml of
cells. (The particle-to-total-cell-ratio is 50:1). Gently swirl the cell/particle mixture
to resuspend the cells. The cells are incubated for 20 minutes at 4° C. Swirl the
cell/particle suspension every 10 minutes to promote attachment. Magnetically separate for
10 minutes and save the supernatant for a negative selection or save the magnetic pellet
for a positive selection.
6. Centrifuge and resuspend the cells in fresh medium for negatively selected cells.
For positively selected cells, refer to "Removal of Cells from BMP After Positive
Selection, Section H.
G. General Procedure for the Direct Method of
Separating Lymphocytes
This procedure is for negative or positive cell selection is best carried out in
sterile tissue culture tubes, or flasks. The following is an example of a one step
protocol which uses BMP covalently attached to CD8 monoclonal antibody (code #
209-318-354). The target population is assumed to be 25% of the total. All steps are done
at 4 ° C. The example uses a particle to total cell ratio of 100:1 for BMP anti-CD8
which is supplied at a concentration of 1 mg/ml.
1. Deliver one ml of approximately 1 x 106 cells in RPMI with 5% fetal
bovine serum and 1% penicillin-streptomycin into an appropriate tube.
2. Wash 0.2 ml of BMP anti-CD8 magnetic particles three times in 0.2 ml of sterile
medium containing antibiotics. Use a magnet to pull the magnetic particles to the side of
the tube and shake vigorously to resuspend the magnetic particles during washing.
3. Add 0.2 ml of washed BMP anti-CD8 magnetic particles to the cells and swirl to mix
the cells with the BMP. (The particle-to-cell-ratio in this example is 100 particles per
cell based on the total cell population).
4. Incubate the cells with the BMP anti-CD8 for 20-30 minutes. Swirl the cell/particle
suspension once every ten minutes to promote attachment. Magnetically separate the
supernatant for 10 minutes twice and save the supernatant for a negative selection or save
the magnetic pellet for a positive selection.
5. Centrifuge and resuspend the negatively selected cells in fresh medium for negative
selection. For positively selected cells, refer to "Removal of Cells from BMP after
Positive Selection, Section H.
H. Removal of Cells from BMP after Positive
Selection
1. Methods for detaching magnetic particles from cells after separation include
culturing cells for up to 48 hours during which magnetic particles fall away from the
cells due to cell surface turnover (See References, Pricop et. al.) or using a protease,
such as chymopapain, to break the antigen-antibody bond. Each of these procedures has
limitations. Not all particles may detach from cells during culturing and the use of a
protease may damage cells. Depending upon the application, it may NOT be necessary
to remove the cells from the BMP. BMP are only 1 µm in size and can be successfully used
in flow cytometry equipment as they will not jam the equipment and are distinguishable
from cells. Alternatively, negative selection should be considered.
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Additional information on BMP and BMP antibody conjugates
is available from our Technical Service staff upon request. Please call 8 a.m. to 5 p.m.
EST, fax or e-mail your request at any time. Access this document from the web at www.rockland-inc.com/morebmpinfo.html.
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