Changes in intracellular calcium concentration and pH of target cells during the cytotoxic process: A quantitative study at the single cell level

BACKGROUND Analysis of chromosomal aberrations or single-gene disorders from rare fetal cells circulating in the blood of pregnant women requires verification of the cells' genomic identity. We have developed a method enabling multiple analyses at the single-cell level that combines verification of the genomic identity of microchimeric cells with molecular genetic and cytogenetic diagnosis. METHODS We used a model system of peripheral blood mononuclear cells spiked with a colon adenocarcinoma cell line and immunofluorescence staining for cytokeratin in combination with DNA staining with the nuclear dye TO-PRO-3 in a preliminary study to define candidate microchimeric (tumor) cells in Cytospin preparations. After laser microdissection, we performed low-volume on-chip isothermal whole-genome amplification (iWGA) of single and pooled cells. RESULTS DNA fingerprint analysis of iWGA aliquots permitted successful identification of all analyzed candidate microchimeric cell preparations (6 samples of pooled cells, 7 samples of single cells). Sequencing of 3 single-nucleotide polymorphisms was successful at the single-cell level for 20 of 32 allelic loci. Metaphase comparative genomic hybridization (mCGH) with iWGA products of single cells showed the gains and losses known to be present in the genomic DNA of the target cells. CONCLUSIONS This method may be instrumental in cell-based noninvasive prenatal diagnosis. Furthermore, the possibility to perform mCGH with amplified DNA from single cells offers a perspective for the analysis of nonmicrochimeric rare cells exhibiting genomic alterations, such as circulating tumor cells.

Download Full-text

Intracellular Calcium Concentration in C6BU-1 Rat Glioma Cells: A Single Cell Study Using Fluorescence Microscopy/Video Imaging System

Psychiatry and Clinical Neurosciences ◽

10.1111/j.1440-1819.1993.tb02132.x ◽

1993 ◽

Vol 47 (2) ◽

pp. 423-423

Author(s):

Takayuki Yamaji ◽

Ariyuki Kagaya ◽

Yasumasa Okamoto ◽

Teruo Hayashi ◽

Ikuo Nagaoka ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Intracellular Calcium ◽

Single Cell ◽

Calcium Concentration ◽

Imaging System ◽

Glioma Cells ◽

Intracellular Calcium Concentration ◽

Video Imaging ◽

Rat Glioma ◽

Cell Study

Download Full-text

Characteristics and requirements of the interaction between human monocytes and tumor cells on the single cell level

Blood ◽

10.1182/blood.v61.2.390.390 ◽

1983 ◽

Vol 61 (2) ◽

pp. 390-396 ◽

Cited By ~ 14

Author(s):

MG Golightly ◽

DG Fischer ◽

C Ohlander ◽

HS Koren

Keyword(s):

Tumor Cell ◽

Single Cell ◽

Tumor Cells ◽

Autologous Serum ◽

Target Cells ◽

Single Cell Level ◽

Human Monocytes ◽

Blood Monocytes ◽

Cell Level ◽

Effector Cells

Abstract Highly purified (97%-99%) and viable (99%) peripheral blood monocytes obtained by EDTA-reversible adherence to autologous-serum-precoated plastic surfaces could rapidly lyse a variety of tumor cells in a 3–4 hr 51Cr release assay. Using these monocytes as effectors, a short-term agarose/conjugate assay was utilized, permitting us to examine the interaction between fresh human monocytes and neoplastic target cells on a single cell level. That the tumor-bound effector cells were indeed monocytes was confirmed by employing the monocyte-specific monoclonal antibody 61D3, which stained 95%-99% of the mononuclear cells bound to conjugated and killed K562 tumor targets. The binding of monocytes to target cells appeared to be temperature dependent and was extremely rapid, reaching a plateau after 5 min at 30 degrees C. Our findings demonstrated for the first time that only a proportion of human blood monocytes can bind to a particular target cell and that only a fraction of the binding cells have the intrinsic potential to kill those neoplastic targets. The proportion of monocytes capable of binding and killing varies between individuals and also depends on the tumor cell used, indicating heterogeneity in the monocyte and tumor cell populations. The highest proportion of monocytes bind to the human erythromyeloid leukemia K562 cell line (13%-50%). The frequency of monocytes capable of killing K562 tumor cells is relatively low (7%- 13%). The system described here should be useful to study the heterogeneity of mononuclear phagocytes and to analyze the molecular basis of the interaction between those effector cells and neoplastic target cells.

Download Full-text

Characteristics and requirements of the interaction between human monocytes and tumor cells on the single cell level

Blood ◽

10.1182/blood.v61.2.390.bloodjournal612390 ◽

1983 ◽

Vol 61 (2) ◽

pp. 390-396

Author(s):

MG Golightly ◽

DG Fischer ◽

C Ohlander ◽

HS Koren

Keyword(s):

Tumor Cell ◽

Single Cell ◽

Tumor Cells ◽

Autologous Serum ◽

Target Cells ◽

Single Cell Level ◽

Human Monocytes ◽

Blood Monocytes ◽

Cell Level ◽

Effector Cells

Highly purified (97%-99%) and viable (99%) peripheral blood monocytes obtained by EDTA-reversible adherence to autologous-serum-precoated plastic surfaces could rapidly lyse a variety of tumor cells in a 3–4 hr 51Cr release assay. Using these monocytes as effectors, a short-term agarose/conjugate assay was utilized, permitting us to examine the interaction between fresh human monocytes and neoplastic target cells on a single cell level. That the tumor-bound effector cells were indeed monocytes was confirmed by employing the monocyte-specific monoclonal antibody 61D3, which stained 95%-99% of the mononuclear cells bound to conjugated and killed K562 tumor targets. The binding of monocytes to target cells appeared to be temperature dependent and was extremely rapid, reaching a plateau after 5 min at 30 degrees C. Our findings demonstrated for the first time that only a proportion of human blood monocytes can bind to a particular target cell and that only a fraction of the binding cells have the intrinsic potential to kill those neoplastic targets. The proportion of monocytes capable of binding and killing varies between individuals and also depends on the tumor cell used, indicating heterogeneity in the monocyte and tumor cell populations. The highest proportion of monocytes bind to the human erythromyeloid leukemia K562 cell line (13%-50%). The frequency of monocytes capable of killing K562 tumor cells is relatively low (7%- 13%). The system described here should be useful to study the heterogeneity of mononuclear phagocytes and to analyze the molecular basis of the interaction between those effector cells and neoplastic target cells.

Download Full-text