J. Cell Sci. 62, 149-159 (1983) 149
Pnnted in Great Britain © The Company of Biologists Limited 1983
CELL FRACTIONATION WITH AFFINITY LIGANDS
CONJUGATED TO AGAROSE-POLYACROLEIN
S. MARGEL*, M. OFARIM AND Z. ESHHARf
The Departments of Plastics Research and f Chemical Immunology, The Weizmann
Institute of Science, Rehovot, Israel
A new effective insoluble support useful for cell fractionation based on agarose-polyacrolein
microsphere beads (APAMB) of diameters 150-250 ^m has been developed. The synthesized
polyacrolein (PA) microspheres, of average diameter 0-2[*m, are provided with reactive aldehyde
groups through which various ligands containing primary amino groups are bound covalently in a
single step at physiological pH. Antibodies coupled to the microspheres are very effective for
labelling of cell surface receptors on human red blood cells and mouse lymphoid cells.
A PAM B were obtained by encapsulating the PA microspheres with agarose. Antibodies and lectins
bound to the APAMB served to construct affinity columns for the separation of red blood cells and
murine lymphocyte subpopulations. Anti-human red blood cell antibodies coupled to anti-
immunoglobulin APAMB are effective in separating human from turkey red blood cells, whereas
either anti-Thy 1-2 anti-immunoglobulin antibodies or soybean agglutinin coupled to APAMB have
proved useful for the separation of T and B cells from heterogeneous population of spleen cells. The
separation procedure is simple, rapid and effective. The viability of the fractionated cells is unaffected
by the procedure and the recovery of the cells is high: between 80 % and 100 %.
The understanding of many biological phenomena, such as the molecular mechan-
ism involved in cell recognition and interaction, cell specialization and malignancy,
requires the development of a reliable method for fractionating various cell sub-
populations. Many fractionation methods based on various properties of cells have
been developed. The main methods include cell electrophoresis (Boehmer, Shortman
& Nossal, 1974), fluorescent sorting (Hulett, Bonner, Sweet & Herzenberg, 1973),
gravity sedimentation (Faguet, 1974; Reisner& Sharon, 1980), glass, nylon and cotton
columns for removal of adherent cells (Julius, Simpson & Herzenberg, 1973), and af-
finity chromatography with various types of beads, e.g. Sepharose (Nicola, Burgess,
Metcalf & Battye, 1978), agarose-polyacrylamide beads (Antoine, Ternynck,
Rodrigot & Avrameas, 1978) and magnetic microspheres (Jovin & Jovin, 1980;
Molday, Yen & Rembaum, 1977).
In this paper the synthesis of a new effective insoluble support for cell fractionation,
consisting of agarose-polyacrolein microsphere beads (APAMB) is described. The
polyacrolein microspheres situated on the surface of the APAMB were used for
covalent binding of appropriate amino ligands in a single step, at physiological pH.
• To whom reprint requests should be directed.
150 S. Margel, M. Ofarim and Z. Eshhar
APAMB with bound ligands were used for specific labelling and, thereby, separation
of the desired cell population. The separation procedure is simple, rapid and effective.
The viability of the fractionated cells is unaffected by the procedure and the recovery
MATERIALS AND METHODS
The following reagents were purchased from commercial sources: goat anti-rabbit immuno-
globulin G (GaRIgG), mouse Ig and poly-L-lysine HBr (Miles Yeda, Rehovot, Israel), rabbit anti-
human red blood cells (RaHRBC) (Cappel Lab. Inc., Cochranville, PA), bovine serum albumin
(BSA, Sigman, St Louis, MO), horse serum (HS) (Bio Lab., Jerusalem, Israel), tetramethyl
rhodamine isothiocyanate (Research Organics Inc., Cleveland, Ohio), fluorescein isothiocyanate
(FITC), polyethylene oxide, M, average 100000 (Polyscience, Warrington, PA), trypan blue
(Fluka, Buchs, Switzerland), D-galactose (BDH, Poole, England), peanut oil (Amend, Irvington,
NJ), agarose A and Sepharosc 4B (Pharmacia Fine Chemicals, Uppsala, Sweden), acrolein,
glutaraldehyde, ethylenediamine, sodium azide, ammonium sulphate and ethanol amine (Aldrich,
Milwakee, WIS). Soybean agglutinin was a kind gift from N. Sharon's group, The Weizmann
Institute of Science, Rehovot, Israel. Acrolein was distilled at atmospheric pressure before use. The
amino derivative of tetramethyl rhodamine isothiocyanate was obtained by dissolving the
tetramethyl rhodamine isothiocyanate in an excess of ethylenediamine in a weight ratio of 1:20.
GaRIgG was purified according to a previously published procedure (Margel, Zisblatt & Rem-
baum, 1979). Monoclonal anti-Thy 1-2 antibodies were obtained by the ammonium sulphate
precipitation of the ascitic fluid obtained from the inoculation of (AKR/J X Balb/c)Fi mice with
the HO-13-4 hybrid cells (Rothstein et al. 1979). Goat anti-mouse Ig (GaMIg) antiserum was
obtained by hyperimmunizing goats with mouse (Fab)2 fragments. Purified antibodies were then
obtained by affinity chromatography on normal mouse Ig coupled to Sepharose 4B. Tetramethyl
rhodamine isothiocyanate conjugated to GaMIg and FITC conjugated to monoclonal anti-Thy 1-2
were obtained according to Brandtzaegl (1973).
Synthesis of poly acrolein microspheres
Polyacrolein (PA) microspheres were prepared by polymerizing acrolein (10%, w/v) in the
presence of polyethylene oxide (0-5 %, w/v) as surfactant with a cobalt radiation source (1 Mrad).
Fluorescent microspheres were obtained by earring out the polymerization in the presence of
0'00S % (w/v) of the amino derivative of tetramethyl rhodamine isothiocyanate. The microspheres
were subsequently extensively dialysed against distilled water and then centrifuged four times at
2000£ for 20 min. The microspheres, of average diameter 0-2fun, as determined by scanning
electron microscopy (SEM), were redispersed in phosphate-buffered saline, 0-01 M, pH 7-2 (PBS)
or in distilled water.
Preparation of cell suspensions
Human red blood cells (RBC) and turkey RBC were obtained by collecting whole normal blood
into tubes containing citrate as anticoagulant. The RBC were washed by centrifugation four times
at 250gfor 7 min at 4°C. After washing the cell pellet was resuspended in PBS or Hanks' solution.
Mouse splenocytes and thymocytes were obtained from female 8- to 10-week old Balb/c mice.
The lymphoid organs were removed and teased into Hanks' solution at 4 °C. After filtration through
nylon gauze the cells were washed four times by successive centrifugation at 350 £ for 10 min at 4 °C.
The cell pellet was then resuspended in PBS or Hanks' solution. Cells were counted in a haemo-
cytometer and their viability determined using trypan blue.
Labelling of human RBC
Rhodaminated PA microspheres were shaken for 2 h at 4°C with purified GaRIgG (1 mg micro-
spheres, 0-1 mg GaRIgG in a total volume of 0'lSml of PBS). Unbound antibody was then
Cell fractionation with immuno-microspheres 151
removed by passing the microsphere suspension through a Sepharose 4B column and monitoring
the separation spectrophotometrically at 280 nm. The free aldehyde groups of the microsphere-
antibody conjugate were quenched by shaking the beads for 4h at 4°C with either BSA solution
(2%, w/v) or with ethanol amine solution brought to pH7-2 with HC1.
A mixture of human and turkey RBC was shaken for 50min at 4°C with RaHRBC (10* cells of
each type with 0'8/ig RaHRBC in 0-1 ml PBS). The cells were then separated and washed four
times by spinning the cell suspension at 500 £ for lOmin. The cell mixture was then shaken at 4CC
for 1 h with the GaRIgG-conjugated microspheres (106 sensitized HRBC, 106 turkey RBC, 1 mg
immuno-microspheres in 0-2ml PBS). The cells were separated from unreacted immuno-
microspheres by centrifugation 3 times at SOO^for lOmin. The cell pellet was resuspended in PBS
and examined by fluorescent light microscopy.
Labelling of mouse splenocytes bearing surface immunoglobulins (B cells)
Rhodaminated microspheres were shaken for 3h at 4°C with GaMIg (1 mg microspheres,
0-05 mg GaMIg in 0-1 ml PBS). The separation of unbound antibody and the quenching of free
aldehyde groups were achieved as described for the labelling of human RBC.
Purified mouse splenocytes (106) were then shaken with the GaMIg-bound microspheres for 1 h
at 4°C. The cells were separated from unreacted immuno-microspheres by spinning three times at
500^ for 15 min at 4CC. The labelled cells were resuspended in PBS and examined by fluorescent
The control used for this experiment consisted of GaMIg-conjugated microspheres and mouse
A solution containing 0-96 g agarose in 16 ml distilled water was heated to 95 °C until the gel
melted into a clear solution. The temperature was then decreased to 70 °C and 8 ml of the PA
microsphere solution (12%, w/v) heated to 70°C were added. The solution was stirred for 30min
and then poured into 100ml of peanut oil, stirred at 300rev./min at 70°C. Ten minutes later, the
solution was cooled with ice. The APAMB formed were purified from the oil by several extractions
with ether. Ether was removed by evaporation. The beads obtained ranged from 50-250fun in
diameter. Fractions containing APAMB with diameters 50-150 j«n and 150-250/im were obtained
by passing the beads through appropriate sieves. APAMB with diameters ranging from 300—
450 ^m were prepared by the same procedure but with a stirring rate of the peanut oil solution of
100 rev./min. The APAMB were stored at 4°C in distilled water containing 0-05 % (w/v) sodium
Binding of polylysine—glutaraldehyde to the APAMB
APAMB (20ml) in 35 ml of distilled