Greenberg P L, Baker S, Link M, Minowada J
Blood. 1985 Jan;65(1):190-7.
We utilized the property of antibody adherence to plastic to separate and obtain enriched fractions of human myeloid (CFU-GM), erythroid (BFU-E) and pluripotent (CFU-GEMM) hemopoietic precursor cells. Nonadherent buoyant human marrow cells coated with mouse anti-human HLA-DR monoclonal antibody (Mc ab), an anti-pan T lymphocyte Mc ab (Leu 1/17F12) or a granulocyte--monocyte-specific Mc ab (MCS2) were incubated on polystyrene Petri plates coated with affinity purified goat anti-mouse immunoglobulin G (IgG). Cells bound to the coated plates and nonbound cells were separately recovered ("panned") by differential elution. Analysis of the nonadherent buoyant marrow cells demonstrated 12% to possess HLA-DR, 6% T, 40% MCS2 antigens on their surface by indirect immunofluorescence (IMF). After panning, 15% +/- 8%, 14% +/- 4% and 8% +/- 6% cells were plate-bound by their respective antibodies, demonstrating differing binding efficiencies. A substantial degree of purity of the recovered cell fractions was shown for bound 74% +/- 6% and 75% +/- 5% IMF positive cells) and nonbound cells (3% +/- 1% and 0.1% +/- 0.8% positive cells) coated with anti-HLA-DR or anti-T Mc ab respectively, with lesser purity for MCS2 panned cells. Seventy-three percent to 126% CFU recovery was noted, with a sevenfold enrichment of the HLA-DR bound cells for CFU-GM and CFU-GEMM, and 3.5-fold enrichment for BFU-E. Sequential panning, obtaining T nonbound-DR bound-surface immunoglobulin nonbound fractions, resulted in tenfold CFU-GM enrichment (107/10(4) cells, approximately equal to 1/100). Anti-MCS2 antibody was ineffective for panning, but use of this antibody in fluorescence-activated cell sorting (FACS) indicated the absence of the MCS2 antigen on the vast majority of CFU-GM. This study describes a relatively rapid and inexpensive method for obtaining enriched antigenically defined hemopoietic precursors in high yield. These techniques should prove useful for more clearly evaluating cellular and humoral interactions with hemopoietic precursor cells.
我们利用抗体与塑料的黏附特性来分离并获得人髓系（CFU - GM）、红系（BFU - E）和多能（CFU - GEMM）造血前体细胞的富集组分。将包被有小鼠抗人HLA - DR单克隆抗体（Mc ab）、抗全T淋巴细胞Mc ab（Leu 1/17F12）或粒细胞 - 单核细胞特异性Mc ab（MCS2）的非黏附性悬浮人骨髓细胞，在包被有亲和纯化山羊抗小鼠免疫球蛋白G（IgG）的聚苯乙烯培养皿上孵育。通过差异洗脱分别回收黏附在包被板上的细胞和未黏附的细胞（“淘选”）。通过间接免疫荧光法（IMF）分析非黏附性悬浮骨髓细胞，结果显示12%的细胞表面有HLA - DR，6%有T抗原，40%有MCS2抗原。淘选后，15%±8%、14%±4%和8%±6%的细胞分别被各自的抗体黏附在板上，表明结合效率不同。对于分别包被有抗HLA - DR或抗T Mc ab的黏附细胞（74%±6%和75%±5%的IMF阳性细胞）和未黏附细胞（3%±1%和0.1%±0.8%的阳性细胞），回收的细胞组分显示出相当程度的纯度，而MCS2淘选细胞的纯度较低。观察到CFU回收率为73%至126%，CFU - GM和CFU - GEMM中与HLA - DR结合的细胞富集了7倍，BFU - E富集了3.5倍。连续淘选，获得T未黏附 - DR黏附 - 表面免疫球蛋白未黏附组分，使CFU - GM富集了10倍（107/10(4)细胞，约等于1/100）。抗MCS2抗体淘选无效，但在荧光激活细胞分选（FACS）中使用该抗体表明绝大多数CFU - GM上不存在MCS2抗原。本研究描述了一种相对快速且廉价的方法，可高产率地获得抗原定义明确的富集造血前体细胞。这些技术应有助于更清楚地评估与造血前体细胞的细胞和体液相互作用。
