- Email: [email protected]
Flow cytometry and cell-separation procedures
Flow cytometry and cell-separation procedures Battye and Shortman
and beads as a slurry ‘in a small sealed tube rather than as a dilute suspension, and diluting to normal suspension volumes before magnetic separation (K Shortman, unpublished data). Some other parameters affecting the efficiencyof separation have been investigated including head to cell ratio [9’], reaction time [6,9*], mixing procedures [6], number of antibodies used [Y], nature of the anti-immunoglobulins linked to the beads [lo] and procedures for coupling antibodies to the beads [6].
Immunomagnetic-bead
separation:
positive
enrichment The immunomagnetic bead approach may also be used for positive selection of cells of interest. Because a small loss of cells can be tolerated, this separation procedure generally requires fewer beads. Two basic problems are encountered in these enrichment procedures. The first is the removal of excess free beads; the use of the small colloidal microspheres offers advantages here, because they can be removed by simple centrifugal washing of the cells. The second problem is the separation of the cells of interest from the beads, and again small microspheres offer the advantage that they can usually be left on the cells without affecting function or subsequent cell sorting [2=*,3=,&l 11. In some circumstances even the largesized beads can be left attached to the cells, for example during the cloning of antigen-specific hybridomas [ 12**], but this is not the general rule. Mechanical disruption of the complexes risks damage to the cells, and although culturing the complexes for a short time sometimes releases the cells [ 131 this is not always effective [PI. h encouraging approach to the release of cells from the larger sized beads has been the use of the proteolytic enzyme chymopapain [ 5**]. Whether this approach is restricted to situations in which there is a conveniently situated, susceptible bond on the target cell-surface antigen, remains to be seen.
Additional
cell-separation
procedures
The specificity of monoclonal antibodies can also be exploited in separation procedures that do not involve the use of paramagnetic beads. Anti-glycophorin-A antibodies show promise in the elimination of erythroidlineage cells from mononuclear cell preparations by aggregation [ 141, an alternative to the usual selective lysis procedures. Columns of avidin-coated polyacrylamide beads (non-magnetic) have been used to separate bone-marrow stem cells coated with anti-CD34 and biotinylated anti-immunoglobulin, with an apparently high rate of recovery of the active stem cells after mechanical disruptions of the column [ 151. The binding of antibod ies to cells may also be used to alter the physical properties of cells thus enabling them to be separated from the other cells. Examples of this procedure are the change in electrophoretic mobility of cells when coated with an-
tibodies [ 161 or the reduction in buoyant density of cells after interaction with antibody-loaded targeted llposomes [171.
Identification
and separation
of stem cells
The rarity of stem cells, and the complexity of the bonemarrow population, are the obvious problems encountered in identifying and separating haemopoietic stem cells. The proposed solutions to these problems in human bone marrow are diverse, ranging from extensive removal of unwanted cells by a combination of both physical procedures and immunomagnetic-bead depletion using a battery of antibodies [ 181, to immediate positive selection of CD34-bearing stem cells by immunomagnetic beads [5**], by avidin-biotin immunoabsorption bead columns [ 151, or by fluorescence-activated cell sorting [ 191. A combination of depletion procedures and immuno-adherence selection for CD7-bearing cells has been used to enrich cell suspension for early T-cell precursors [20]. However, the experience with murine haemopoietic stem cells suggests that further purification steps will be required and the stem cells will need to be more tightly defined. A recent review of experiments carried out to separate stem cells from haemopoietic tissue in the mouse [ 211, cites work suggesting that the very earliest ‘stem cells for stem cells’ may be a less activated or quiescent subset of the cells phenotypically identified as spleen colony form ing units (CFU-s). A report of work on rat bone marrow cells [22*] describes the capacity of flow cytometry to identify, by three-colour immunofluorescence, a subpopulation of these cells that are 46%fold enriched for CFU-s and simultaneously assess levels of activation. This can be achieved by fluorescent staining of total cellular DNA to show cell-cycle status, by measurement of intracellular Ca2+ levels using the fluorochrome Indo-1, or by use of the mitochondrial-selective dye Rhodamine 123 (where low mitochondrial content implies quiescence). Thus, heterogeneity has been revealed in a population of apparently phenotypically identical cells. Similarly, a subpopulation of cells has been sorted from mouse bone marrow cells by the combination of their strong binding to wheat germ agglutinin and low uptake of the Hoechst 33342 DNA dye [23*]. The resultant cells have been assayed for spleen colony formation and for their ability to reconstitute the haemopoietic system of lethally irradiated mice.
Molecular
biology
readout
in flow cytometry
One of the events required for equipping lymphoid cells for antigen recognition is the rearrangement and assembly of the antigen receptor’s V, D and J germ-line gene segments. Although the enzymes responsible for the rearrangement are not well characterized, VDJ recombinase activity has now been detected by flow cytome-
239
240
Immunological techniques try [24**]. A recombination substrate containing V and J segments together with the kacZ gene was inserted into viable! cells. This substrate was constructed so that inversional rearrangement by means of VDJ recombinase activity was necessary for the ~x.Z’gene to be expressed as P-galactosidase activity. If that happened, fluorescein di-p-D-galactopyranoside, which had also been introduced into the cells, was enzymatically cleaved to fluorescein, which was detectable in flow cytometry. Thus, a key event in lymphocyte development can be monitored at the molecular biological level using flow cytometry.
immunology is a series of synthesized cyanine dyes, converted to active esters (absorption maxima wavelengths 550-750 nm) that should be useful in labelling antibodies or other proteins [30*].
References
application
of flow cytometry
There can be no question that the spread of the acquired Immune deficiency syndrome has hastened the introduction of flow cytometry into the clinical field, where there is intense interest in the enumeration of peripheral-blood cell subpopulations. Recent developments are in two directions. In the first, the investigation of peripheral blood subpopulations has been undertaken at a more sophisticated level using four-colour immunofluorescence. In one study [25-l, natural killer (NK) cells, which were defined as CD16+ and CD56+, were further subdivided into six populations that were assayed separately for NK cell activity. In other work [ 26.1, quantitative differences have been observed in the relative proportions of T-cell subsets, subdivided by four-colour immunofluorescence using CD4, CD& CD45R, CDw29, CD38 and HIA markers, among blood cells obtained from HIV+ or HIV- individuals. The second direction is towards standardization. With the rapid increase in the number of laboratories operating flow-cytometry analyses, a plethora of protocols has developed. In two groups of laboratories [27-,28-l attempts have been made to standardize the procedures so that equivalent results can be obtained at any site within the group. It can be argued that similar standardization should be sought in a global context to enable comparison of flow cytometry results from all research groups, particularly in surface-antigen analysis.
UCELSIXD J, REMBAUM A, KEMSHEAD JT, NUSTADK, FUNDERUD S, SCHMID R: Preparation and Biomedical Applications of Monodisperse Polymer Particles. In MicqOberes and Drug Therapy, Pharmaceutical and Medical A.pect.s edited by Davies SS, Illurn L, McVie JC, l&son T. Amsterdam: Elsevier, 19% pp 365-382.
MILTEZNM S, MULLFZR W, WEICHELA, RADBRUCHA: High Gradient Magnetic Cell Separation with MACS. Cytovze0-y 1’290, 11:231-239. The basic description of the MACS system now available commercially. A user-friendly version of the principle of using small biotinylated paramagnetic particles fo bind fo cells coated with antibody then employing a high gradient magnetic field to separate the bound cells. 2. ..
ABTS H, EMMERICHM, MILTENYIS, RADBRUCK A, TEACHH: CD20 Positive Human B Lymphocytes Separated with the Magnetic Cell Sorter (WCS) can be Indiced to Proliferation and Antibody Secretion In Vitro. J Immunol Methods 1989, 125:19-28. A report of the successive depletion of T cells then enrichment of B cells using MACS; 97% purity and recovery of B-cell function in culture. 3. .
4.
YAU JC, READING CL, THOMAS MW, DAVARAJBM, TINDLE SE, JAGANNA~ S, DICKE KA: Purging of T-lymphocytes with Mag netic AIlInity CoIIoid. Eap Hematol 1990, l&21+222.
CMN CI, STRAUSSLC, FACKLERMJ, TRI~CHMANN TM, WILEYJM, LOKEN MR: Positive Stem Cell Selection - Basic Science. Prog Clin Biol Res 199Q 333:387-402. Positive selection of bone marrow stem cells using anti-CD34 antibody and anti-immunoglobulin-coated Dynabeads. A good rate of recovery and enrichment of functional activity is achieved by using a proteolytic enzyme to separate beads from cells. 5. ..
HEBELLT, GOTZE 0: The Isolation of B Lymphocytes from Human Peripheral Blood Using Antibodies Coupled to Paramagnetic Particles and Rosetting Techniques. J Immunol Methods 1989, 123:28+291. MORGAN4 BURKE LM: Comparison of Mitogen Responses of Lymphocyte Subpopulations Separated Using Coated Metal Beads and by Traditional Methods. J Immunol Met&x& 1990, 126:lA.
New dyes for flow cytometry The next foreseeable development in flow cytometry should be the replacement of unwieldy and powerinefficient ion and dye-laser systems with solid-state lasers. This can only come about when there is overlap of the range of excitation wavelengths of flow cytometry fluorochromes with the available laser wavelengths. Of interest in this context are two reports of new fluorescent dyes. Six dyes selected from the wide range in use in the textile industry were shown to be vital stains, specific for a variety of cell structures and having several excitation wavelengths [29]. Possibly of more interest in
reading
Papers of special interest, published within the annual period of review, have been highlighted as: . of interest .. of outstanding interest 1.
Clinical
and recommended
R, DOPFER R, HAMMERS, IANG P, DAURERB, KIMMIGA, SIED~ERR, JAUXY B, TREUNERJ, NEITHAMME RD:The Efficiency of a New CeII Separation Technique in Bone Marrow Purging Without Damaging the Purged Tumor Ceils. Prog Clin Biol Res 1990, 333:303-309. HANDCRETINGER
PILLINGD, KITAS GD, SALMONM, BACON PA: The Kinetics of Interaction Between Lymphocytes and Magnetic Polymer particles. J Immunol Methb 1989, 122:23>241. A study of the effect of some relevant parameters (bead to cell IX tio, incubation time and simultaneous use of two antibodies) in the separation of antibody-coated cells with anti-immunoglobulin-coated Dynabeads. 10.
WE, JOHNSON KS, LEE C, CASSANOW: Relative Efficiency of Leukemic CeII Depletion Using Anti-murhte-
JAN~~EN
Flow cytometry and cell-separation procedures Battye and Shortman IgGl (Fc) or Anti-murine-IgG Coated Immunomagnetic crobeads. Bone Marrow Trans 1990, 5:1’+22. 11.
Mi-
SCHRODER U, PERO RW, SJ~GRENH-O: Magnetically
Tagged Subsets of Human Lymphocytes for Assays with Laboratory Robotics. Cl&z Cbem 1990, 36:1282-1287.
HORTON JK, EVANSOM, SWANNK, SWINBURNES: A New and Rapid Method for the Selection and Cloning of Antigen-specilic Hybrldomas with Magnetic Microspheres. J Immunol Methods 1989, 124:22%230. In contrast to usual approaches this uses antigen (rather than antibody) coupled to Dynabeads to enrich for antibody-secreting hybridomas. The selected cells are cloned directly, with the beads still bound. This may be useful in the routine production of hybridomas 12. ..
13.
LUNDIN KEA, OV~GSTAD E, SOLUD LM, GJER~SEN m GAUDERNACK G, THORSBY E: Positive Selection of Tat-(CD25) Positive Cells Following T-Cell Activation. Use of Immunomagnetic Separation and Implications for T-Cell Cloning. J Immunogenetics 1989, 16:18>191.
14.
HELDRUP J: A New Technique
Using an Aggregating Antibody Against Glycophorin-A for Purging Ficoll-Paque-separated Leucocytes of Contaminating Erythroid Lineage Cells. Stand J Immunol 1990, 31~2892%.
15.
BERENSON RJ, BESINGER WI, HILLR, ANDREWSRG, GARCLQLOPEZ J, KAIAMsz DF, STIU BJ, BUCKNERCD, BERNSTEINID, THOMAS ED: Stem Cell Selection - Clinical Experience. Prog Clin Biol Res 1990, 333:40%413.
16.
HANSENE, WUSTROWTPU, HANNIG K: Antigen-specific
trophoretic Cell Separation for Immunological tions. Electr@oresis 1989, 10:645-652. 17.
ElecInvestiga-
G~L~EV A, SUOROVAE: The Use of Targeted
Isolate Cells Beating Immunoglobulin Metho& 1989, 125:29-34.
Liposomes to Receptors. J Immunol
18.
COWR-FOX
M, BAZARIs, DEEGHJ: Isolation of Hemopoietic Precursor Cells from Human Marrow by Negative Selection Using Monoclonal Antibodies and Immunomagnetic Beads. Prog Clin Rio1 Res 1990, 333:277-284.
19.
EMA H, SUDA T, MKJRAY, NAKAUCHIH: Colony Formation of Clone-sorted Human Hematopoietic Progenitors. Blood 1990, 75:1941-1946.
20.
BERTHOJM, MOSSALAYI MD, DALUXJLAH, MOUT%XDEG, DEBRE
P: Isolation of Early T-Cell Precursor (CFU-TL) from Human Bone Marrow. Blood 1990, 75:1064-1068. 21.
SPANGRUDE GJ: Enrichment
CelIs: Diverging
Roads.
of Murine Haemopoietic Stem Immunol Toady 1989, 10:344-350.
22. MCCARTHYKF, I-1ALEML: Further Enrichment and Analysis . of Rat CFIJ-s. Int J Cell Cloning 1990, 8:184-195. This paper reports Uow cytometric and lluorescence-activated cell soning techniques for simultaneous phenotypic identification of CFU-s, using three-color immunofluorescence. It also describes flow cytometric analysis of their level of activation by fluorescent staining of DN& by measuring intracellular Ca2 + or by the use of mitochondriaselective dye.
23. .
MCAIXXERI, WOLF NS, PIE’IXZYKME, RABINO~ITCHPS, PRIESTLEY G, JAEGER B: Transplantation of Hematopoietic Stem Cells
Obtained by a Combined Dye Method Fractionation of Murine Bone Marrow. Blood 1990, 75:124&1246. Stem cells were sorted according to their high binding to wheat germ agglutinln and low uptake of Hoechst DNA strain. 24. ..
YANCOPOULOSGD, NOLAN GP, POLLOCKR, PROCKOP S, LI SC, HERZENBERGLA, ALT m A Novel Fluorescence-based System for Assaying and Separating Live Cells According to VDJ RecombActivity. Mol Cell Biol 1990, I0:1697-1704. A specially constructed recombination substrate is inserted into viable cells. Inversional rearrangement of this substrate in each ceU with VDJ recombinase activity leads to fluorescent marking of the cell. Thus, genetic rearrangements in early lymphocyte development can be detected directly by flow cytometry. SROUR EF, LEEMHUIS T, JENSKI L, REDMOND R, JANSEN J: Cytolytic Activity of Human Natural KllIer Ceil Subpopulations Isolated by Four-color Immunofluorescence Flow Cytometric Cell Sorting. Cytometry 1990, 11:442-446. NK cells initially defined by CD16 and CD56 are further subdivided into six groups, each of which is sorted and assayed for NK cell activity 25. .
26. .
SCHNIZLEIN-BICK CT, MAGIER MR, JONES RB, FIFE KH, KATZ BP, WALKEREB: Differences Among Mononuclear Cell Subpop ulations in HIV Scropositive or Seronegative Homosexual and Heterosexual Men as Determined by Four-color Flow Cytometry. J Acquired Immune Deficiency Syndromes 1990,
3:747-756. Subsets of CD4positive or CD8-positive cells are dehned and enumerated using CD45R, CDw29, CD38 and HLA surface markers. PARKER JW, ADEISBERG B, AZEN SP, BOONE D, FLETCHERMA, GJER~ET GF, HASETI J, KAPLANJ, NI~ANDJC, ODOM-MARYON T, ET AL.: Leukocyte Immunophenotyping by Flow Cytometry in a Multisite Study: Standardization, Quality Control, and Normal Values in the Transfusion Safety Study. Clin Immunol Immunopatbol 1990, 55:187-220. A program of quality control and standardization of blood leukocyte im munophenotyping between six geographically separated laboratories. Extensive data on normal values is included.
27. .
28. .
GIORGI JV, CHENG H-L, MARGOUCKJB, BAUER KD, FERBA~ J, WAXDAL M, SCHMID I, HULTIN LE, JACKSON AL, PARK L, TAYLQR JMG Er Ar: Quality Control in the Flow Cytometric Measurement of T-lymphocyte Subsets: the Multicenter AIDS Cohort Study Experience. Clin Immunol Immuncpatbol 1990, 55:173-186. Four flow ojtometry laboratories report their standardization procedures for T-lymphocyte subset analysis. SIMMONSDM, MERCERAV, HALI& G, DYSON JED: Characterization of Six Textile Dyes as Fluorescent Stains for Flow Cytometry. J Histocbem Cytocbem 1990, 38:4149. Selected dyes show specificity for various cell structures and sweral tiferent excitation wavelengths.
29. .
SOU~CK PL, ERNSTLA, TAURIEUX)EW, PARKERSR, MUJUMDAR RB, MUJUMDARSR, CLEVERHk WAGGONERAS: Cyanine Dye Labelling Reagents Carboxymethylindocyanine Succinimidyl Esters. Cytometry 1990, 11:418430. Ten cyanine dyes have been synthesized in forms suitable for antibody or other protein conjugation. Their excitation wavelengths range from 575 nm to 780 nm. 30. .
FL Battye and K Shortman, The Walter and EUza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia.
241
and beads as a slurry ‘in a small sealed tube rather than as a dilute suspension, and diluting to normal suspension volumes before magnetic separation (K Shortman, unpublished data). Some other parameters affecting the efficiencyof separation have been investigated including head to cell ratio [9’], reaction time [6,9*], mixing procedures [6], number of antibodies used [Y], nature of the anti-immunoglobulins linked to the beads [lo] and procedures for coupling antibodies to the beads [6].
Immunomagnetic-bead
separation:
positive
enrichment The immunomagnetic bead approach may also be used for positive selection of cells of interest. Because a small loss of cells can be tolerated, this separation procedure generally requires fewer beads. Two basic problems are encountered in these enrichment procedures. The first is the removal of excess free beads; the use of the small colloidal microspheres offers advantages here, because they can be removed by simple centrifugal washing of the cells. The second problem is the separation of the cells of interest from the beads, and again small microspheres offer the advantage that they can usually be left on the cells without affecting function or subsequent cell sorting [2=*,3=,&l 11. In some circumstances even the largesized beads can be left attached to the cells, for example during the cloning of antigen-specific hybridomas [ 12**], but this is not the general rule. Mechanical disruption of the complexes risks damage to the cells, and although culturing the complexes for a short time sometimes releases the cells [ 131 this is not always effective [PI. h encouraging approach to the release of cells from the larger sized beads has been the use of the proteolytic enzyme chymopapain [ 5**]. Whether this approach is restricted to situations in which there is a conveniently situated, susceptible bond on the target cell-surface antigen, remains to be seen.
Additional
cell-separation
procedures
The specificity of monoclonal antibodies can also be exploited in separation procedures that do not involve the use of paramagnetic beads. Anti-glycophorin-A antibodies show promise in the elimination of erythroidlineage cells from mononuclear cell preparations by aggregation [ 141, an alternative to the usual selective lysis procedures. Columns of avidin-coated polyacrylamide beads (non-magnetic) have been used to separate bone-marrow stem cells coated with anti-CD34 and biotinylated anti-immunoglobulin, with an apparently high rate of recovery of the active stem cells after mechanical disruptions of the column [ 151. The binding of antibod ies to cells may also be used to alter the physical properties of cells thus enabling them to be separated from the other cells. Examples of this procedure are the change in electrophoretic mobility of cells when coated with an-
tibodies [ 161 or the reduction in buoyant density of cells after interaction with antibody-loaded targeted llposomes [171.
Identification
and separation
of stem cells
The rarity of stem cells, and the complexity of the bonemarrow population, are the obvious problems encountered in identifying and separating haemopoietic stem cells. The proposed solutions to these problems in human bone marrow are diverse, ranging from extensive removal of unwanted cells by a combination of both physical procedures and immunomagnetic-bead depletion using a battery of antibodies [ 181, to immediate positive selection of CD34-bearing stem cells by immunomagnetic beads [5**], by avidin-biotin immunoabsorption bead columns [ 151, or by fluorescence-activated cell sorting [ 191. A combination of depletion procedures and immuno-adherence selection for CD7-bearing cells has been used to enrich cell suspension for early T-cell precursors [20]. However, the experience with murine haemopoietic stem cells suggests that further purification steps will be required and the stem cells will need to be more tightly defined. A recent review of experiments carried out to separate stem cells from haemopoietic tissue in the mouse [ 211, cites work suggesting that the very earliest ‘stem cells for stem cells’ may be a less activated or quiescent subset of the cells phenotypically identified as spleen colony form ing units (CFU-s). A report of work on rat bone marrow cells [22*] describes the capacity of flow cytometry to identify, by three-colour immunofluorescence, a subpopulation of these cells that are 46%fold enriched for CFU-s and simultaneously assess levels of activation. This can be achieved by fluorescent staining of total cellular DNA to show cell-cycle status, by measurement of intracellular Ca2+ levels using the fluorochrome Indo-1, or by use of the mitochondrial-selective dye Rhodamine 123 (where low mitochondrial content implies quiescence). Thus, heterogeneity has been revealed in a population of apparently phenotypically identical cells. Similarly, a subpopulation of cells has been sorted from mouse bone marrow cells by the combination of their strong binding to wheat germ agglutinin and low uptake of the Hoechst 33342 DNA dye [23*]. The resultant cells have been assayed for spleen colony formation and for their ability to reconstitute the haemopoietic system of lethally irradiated mice.
Molecular
biology
readout
in flow cytometry
One of the events required for equipping lymphoid cells for antigen recognition is the rearrangement and assembly of the antigen receptor’s V, D and J germ-line gene segments. Although the enzymes responsible for the rearrangement are not well characterized, VDJ recombinase activity has now been detected by flow cytome-
239
240
Immunological techniques try [24**]. A recombination substrate containing V and J segments together with the kacZ gene was inserted into viable! cells. This substrate was constructed so that inversional rearrangement by means of VDJ recombinase activity was necessary for the ~x.Z’gene to be expressed as P-galactosidase activity. If that happened, fluorescein di-p-D-galactopyranoside, which had also been introduced into the cells, was enzymatically cleaved to fluorescein, which was detectable in flow cytometry. Thus, a key event in lymphocyte development can be monitored at the molecular biological level using flow cytometry.
immunology is a series of synthesized cyanine dyes, converted to active esters (absorption maxima wavelengths 550-750 nm) that should be useful in labelling antibodies or other proteins [30*].
References
application
of flow cytometry
There can be no question that the spread of the acquired Immune deficiency syndrome has hastened the introduction of flow cytometry into the clinical field, where there is intense interest in the enumeration of peripheral-blood cell subpopulations. Recent developments are in two directions. In the first, the investigation of peripheral blood subpopulations has been undertaken at a more sophisticated level using four-colour immunofluorescence. In one study [25-l, natural killer (NK) cells, which were defined as CD16+ and CD56+, were further subdivided into six populations that were assayed separately for NK cell activity. In other work [ 26.1, quantitative differences have been observed in the relative proportions of T-cell subsets, subdivided by four-colour immunofluorescence using CD4, CD& CD45R, CDw29, CD38 and HIA markers, among blood cells obtained from HIV+ or HIV- individuals. The second direction is towards standardization. With the rapid increase in the number of laboratories operating flow-cytometry analyses, a plethora of protocols has developed. In two groups of laboratories [27-,28-l attempts have been made to standardize the procedures so that equivalent results can be obtained at any site within the group. It can be argued that similar standardization should be sought in a global context to enable comparison of flow cytometry results from all research groups, particularly in surface-antigen analysis.
UCELSIXD J, REMBAUM A, KEMSHEAD JT, NUSTADK, FUNDERUD S, SCHMID R: Preparation and Biomedical Applications of Monodisperse Polymer Particles. In MicqOberes and Drug Therapy, Pharmaceutical and Medical A.pect.s edited by Davies SS, Illurn L, McVie JC, l&son T. Amsterdam: Elsevier, 19% pp 365-382.
MILTEZNM S, MULLFZR W, WEICHELA, RADBRUCHA: High Gradient Magnetic Cell Separation with MACS. Cytovze0-y 1’290, 11:231-239. The basic description of the MACS system now available commercially. A user-friendly version of the principle of using small biotinylated paramagnetic particles fo bind fo cells coated with antibody then employing a high gradient magnetic field to separate the bound cells. 2. ..
ABTS H, EMMERICHM, MILTENYIS, RADBRUCK A, TEACHH: CD20 Positive Human B Lymphocytes Separated with the Magnetic Cell Sorter (WCS) can be Indiced to Proliferation and Antibody Secretion In Vitro. J Immunol Methods 1989, 125:19-28. A report of the successive depletion of T cells then enrichment of B cells using MACS; 97% purity and recovery of B-cell function in culture. 3. .
4.
YAU JC, READING CL, THOMAS MW, DAVARAJBM, TINDLE SE, JAGANNA~ S, DICKE KA: Purging of T-lymphocytes with Mag netic AIlInity CoIIoid. Eap Hematol 1990, l&21+222.
CMN CI, STRAUSSLC, FACKLERMJ, TRI~CHMANN TM, WILEYJM, LOKEN MR: Positive Stem Cell Selection - Basic Science. Prog Clin Biol Res 199Q 333:387-402. Positive selection of bone marrow stem cells using anti-CD34 antibody and anti-immunoglobulin-coated Dynabeads. A good rate of recovery and enrichment of functional activity is achieved by using a proteolytic enzyme to separate beads from cells. 5. ..
HEBELLT, GOTZE 0: The Isolation of B Lymphocytes from Human Peripheral Blood Using Antibodies Coupled to Paramagnetic Particles and Rosetting Techniques. J Immunol Methods 1989, 123:28+291. MORGAN4 BURKE LM: Comparison of Mitogen Responses of Lymphocyte Subpopulations Separated Using Coated Metal Beads and by Traditional Methods. J Immunol Met&x& 1990, 126:lA.
New dyes for flow cytometry The next foreseeable development in flow cytometry should be the replacement of unwieldy and powerinefficient ion and dye-laser systems with solid-state lasers. This can only come about when there is overlap of the range of excitation wavelengths of flow cytometry fluorochromes with the available laser wavelengths. Of interest in this context are two reports of new fluorescent dyes. Six dyes selected from the wide range in use in the textile industry were shown to be vital stains, specific for a variety of cell structures and having several excitation wavelengths [29]. Possibly of more interest in
reading
Papers of special interest, published within the annual period of review, have been highlighted as: . of interest .. of outstanding interest 1.
Clinical
and recommended
R, DOPFER R, HAMMERS, IANG P, DAURERB, KIMMIGA, SIED~ERR, JAUXY B, TREUNERJ, NEITHAMME RD:The Efficiency of a New CeII Separation Technique in Bone Marrow Purging Without Damaging the Purged Tumor Ceils. Prog Clin Biol Res 1990, 333:303-309. HANDCRETINGER
PILLINGD, KITAS GD, SALMONM, BACON PA: The Kinetics of Interaction Between Lymphocytes and Magnetic Polymer particles. J Immunol Methb 1989, 122:23>241. A study of the effect of some relevant parameters (bead to cell IX tio, incubation time and simultaneous use of two antibodies) in the separation of antibody-coated cells with anti-immunoglobulin-coated Dynabeads. 10.
WE, JOHNSON KS, LEE C, CASSANOW: Relative Efficiency of Leukemic CeII Depletion Using Anti-murhte-
JAN~~EN
Flow cytometry and cell-separation procedures Battye and Shortman IgGl (Fc) or Anti-murine-IgG Coated Immunomagnetic crobeads. Bone Marrow Trans 1990, 5:1’+22. 11.
Mi-
SCHRODER U, PERO RW, SJ~GRENH-O: Magnetically
Tagged Subsets of Human Lymphocytes for Assays with Laboratory Robotics. Cl&z Cbem 1990, 36:1282-1287.
HORTON JK, EVANSOM, SWANNK, SWINBURNES: A New and Rapid Method for the Selection and Cloning of Antigen-specilic Hybrldomas with Magnetic Microspheres. J Immunol Methods 1989, 124:22%230. In contrast to usual approaches this uses antigen (rather than antibody) coupled to Dynabeads to enrich for antibody-secreting hybridomas. The selected cells are cloned directly, with the beads still bound. This may be useful in the routine production of hybridomas 12. ..
13.
LUNDIN KEA, OV~GSTAD E, SOLUD LM, GJER~SEN m GAUDERNACK G, THORSBY E: Positive Selection of Tat-(CD25) Positive Cells Following T-Cell Activation. Use of Immunomagnetic Separation and Implications for T-Cell Cloning. J Immunogenetics 1989, 16:18>191.
14.
HELDRUP J: A New Technique
Using an Aggregating Antibody Against Glycophorin-A for Purging Ficoll-Paque-separated Leucocytes of Contaminating Erythroid Lineage Cells. Stand J Immunol 1990, 31~2892%.
15.
BERENSON RJ, BESINGER WI, HILLR, ANDREWSRG, GARCLQLOPEZ J, KAIAMsz DF, STIU BJ, BUCKNERCD, BERNSTEINID, THOMAS ED: Stem Cell Selection - Clinical Experience. Prog Clin Biol Res 1990, 333:40%413.
16.
HANSENE, WUSTROWTPU, HANNIG K: Antigen-specific
trophoretic Cell Separation for Immunological tions. Electr@oresis 1989, 10:645-652. 17.
ElecInvestiga-
G~L~EV A, SUOROVAE: The Use of Targeted
Isolate Cells Beating Immunoglobulin Metho& 1989, 125:29-34.
Liposomes to Receptors. J Immunol
18.
COWR-FOX
M, BAZARIs, DEEGHJ: Isolation of Hemopoietic Precursor Cells from Human Marrow by Negative Selection Using Monoclonal Antibodies and Immunomagnetic Beads. Prog Clin Rio1 Res 1990, 333:277-284.
19.
EMA H, SUDA T, MKJRAY, NAKAUCHIH: Colony Formation of Clone-sorted Human Hematopoietic Progenitors. Blood 1990, 75:1941-1946.
20.
BERTHOJM, MOSSALAYI MD, DALUXJLAH, MOUT%XDEG, DEBRE
P: Isolation of Early T-Cell Precursor (CFU-TL) from Human Bone Marrow. Blood 1990, 75:1064-1068. 21.
SPANGRUDE GJ: Enrichment
CelIs: Diverging
Roads.
of Murine Haemopoietic Stem Immunol Toady 1989, 10:344-350.
22. MCCARTHYKF, I-1ALEML: Further Enrichment and Analysis . of Rat CFIJ-s. Int J Cell Cloning 1990, 8:184-195. This paper reports Uow cytometric and lluorescence-activated cell soning techniques for simultaneous phenotypic identification of CFU-s, using three-color immunofluorescence. It also describes flow cytometric analysis of their level of activation by fluorescent staining of DN& by measuring intracellular Ca2 + or by the use of mitochondriaselective dye.
23. .
MCAIXXERI, WOLF NS, PIE’IXZYKME, RABINO~ITCHPS, PRIESTLEY G, JAEGER B: Transplantation of Hematopoietic Stem Cells
Obtained by a Combined Dye Method Fractionation of Murine Bone Marrow. Blood 1990, 75:124&1246. Stem cells were sorted according to their high binding to wheat germ agglutinln and low uptake of Hoechst DNA strain. 24. ..
YANCOPOULOSGD, NOLAN GP, POLLOCKR, PROCKOP S, LI SC, HERZENBERGLA, ALT m A Novel Fluorescence-based System for Assaying and Separating Live Cells According to VDJ RecombActivity. Mol Cell Biol 1990, I0:1697-1704. A specially constructed recombination substrate is inserted into viable cells. Inversional rearrangement of this substrate in each ceU with VDJ recombinase activity leads to fluorescent marking of the cell. Thus, genetic rearrangements in early lymphocyte development can be detected directly by flow cytometry. SROUR EF, LEEMHUIS T, JENSKI L, REDMOND R, JANSEN J: Cytolytic Activity of Human Natural KllIer Ceil Subpopulations Isolated by Four-color Immunofluorescence Flow Cytometric Cell Sorting. Cytometry 1990, 11:442-446. NK cells initially defined by CD16 and CD56 are further subdivided into six groups, each of which is sorted and assayed for NK cell activity 25. .
26. .
SCHNIZLEIN-BICK CT, MAGIER MR, JONES RB, FIFE KH, KATZ BP, WALKEREB: Differences Among Mononuclear Cell Subpop ulations in HIV Scropositive or Seronegative Homosexual and Heterosexual Men as Determined by Four-color Flow Cytometry. J Acquired Immune Deficiency Syndromes 1990,
3:747-756. Subsets of CD4positive or CD8-positive cells are dehned and enumerated using CD45R, CDw29, CD38 and HLA surface markers. PARKER JW, ADEISBERG B, AZEN SP, BOONE D, FLETCHERMA, GJER~ET GF, HASETI J, KAPLANJ, NI~ANDJC, ODOM-MARYON T, ET AL.: Leukocyte Immunophenotyping by Flow Cytometry in a Multisite Study: Standardization, Quality Control, and Normal Values in the Transfusion Safety Study. Clin Immunol Immunopatbol 1990, 55:187-220. A program of quality control and standardization of blood leukocyte im munophenotyping between six geographically separated laboratories. Extensive data on normal values is included.
27. .
28. .
GIORGI JV, CHENG H-L, MARGOUCKJB, BAUER KD, FERBA~ J, WAXDAL M, SCHMID I, HULTIN LE, JACKSON AL, PARK L, TAYLQR JMG Er Ar: Quality Control in the Flow Cytometric Measurement of T-lymphocyte Subsets: the Multicenter AIDS Cohort Study Experience. Clin Immunol Immuncpatbol 1990, 55:173-186. Four flow ojtometry laboratories report their standardization procedures for T-lymphocyte subset analysis. SIMMONSDM, MERCERAV, HALI& G, DYSON JED: Characterization of Six Textile Dyes as Fluorescent Stains for Flow Cytometry. J Histocbem Cytocbem 1990, 38:4149. Selected dyes show specificity for various cell structures and sweral tiferent excitation wavelengths.
29. .
SOU~CK PL, ERNSTLA, TAURIEUX)EW, PARKERSR, MUJUMDAR RB, MUJUMDARSR, CLEVERHk WAGGONERAS: Cyanine Dye Labelling Reagents Carboxymethylindocyanine Succinimidyl Esters. Cytometry 1990, 11:418430. Ten cyanine dyes have been synthesized in forms suitable for antibody or other protein conjugation. Their excitation wavelengths range from 575 nm to 780 nm. 30. .
FL Battye and K Shortman, The Walter and EUza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia.
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