Strategies for cell permeabilization and fixation in detecting surface and intracellular antigens.
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Antibodies↗
Biomedical subjects
W E Bolton
Publications and source records attributed to W E Bolton.
Intracellular markers.
An increased level of complexity will be encountered when developing protocols for intracellular markers. Protocols for surface markers have been successfully standardized, however it is understood that no single method is appropriate for all intracellular staining. A systematic approach should be followed, including knowledge of antigen location and functional state, selection of cell fixative and cell permeabilizer, antibody specificity and class/subclass, fluorochrome, fluorochrome to protein ratio (F:P), and use of adequate controls, including isotype-matched negative controls and positive and negative cell controls. Even though it is impossible to recommend a single technique to stain all intracellular antigens, the authors present a logical approach to follow when developing a staining protocol.
Antibodies↗
Use of artificial neural networks in evaluating prognostic factors determining the response to dendritic cells pulsed with PSMA peptides in prostate cancer patients.
BACKGROUND: Our purpose was to compare the importance of over 22 measurements used in evaluating the clinical responses of patients with metastatic or locally recurrent prostate cancer, treated by dendritic cell (DC) infusions with prostate-specific membrane antigen (PSMA) peptides. METHODS: Artificial neural networks (ANNs) were employed for assessment, as well as the traditional methods of logistic regression. RESULTS: Twenty-six patients with metastatic disease and 37 patients with local recurrence were available for evaluation and comparison. ANN evaluation ranked the collective effects of DC infusion, immune responses (CD3+ cells, CD16+ cells, zeta chain+ cells), and cytokines, e.g., IL-6 and PSMA levels, very highly. Logistic regression identified all of these parameters to some degree, but in a different rank order. Patients with metastases showed a sharp rate of response secondary to the level of DC infusion, in contrast to those patients with local recurrence, in which it was more gradual. CONCLUSIONS: ANN analysis emphasizes the importance of level of DC infusion, immune parameters, cytokines, and markers such as PSMA in determining the response to PSMA peptide immunotherapy. The criteria of response were judged to be correct in 86% of metastatic patients and 83% of locally recurrent patients evaluated in this study.
Clinical Trials, Phase II as Topic↗
Simultaneous detection of cyclin B1, p105, and DNA content provides complete cell cycle phase fraction analysis of cells that endoreduplicate.
BACKGROUND: DNA analysis of endoreduplicating cells is difficult because of the overlap between stem-line G2 + M cells and 4C G1 cells. Simultaneous flow cytometry of DNA and cyclin B1 analytically separates these populations. The objective here was to develop simultaneous flow cytometry of DNA, cyclin B1, and p105 (highly expressed in mitosis) for improved, complete cell cycle phase fraction analysis of endoreduplicating cell populations. METHODS: Monoclonal antibody, GNS-1, reactive with human cyclin B1, was conjugated with fluorescein at three different fluorochrome-to-protein (F/P) ratios and tested for optimal sensitivity in a flow cytometric assay. A formaldehyde-methanol fixation procedure was optimized for retention of p105 within mitotic cells by analytic titration of formaldehyde. p105 was stained indirectly with Cy5-conjugated secondary antibody, followed by GNS-1, and DNA was stained with Hoechst 33342. The specificity of p105 in this assay was tested by comparison of manual and flow cytometric mitotic indices and by sorting and microscopic inspection. RESULTS: F/P 4.1 provided optimal fluorescein labeling of GNS-1. Formaldehyde (0.5%), followed by methanol permeabilization, fixed cells sufficiently to quantify stem-line and endoreduplicated G1, S, G2, and M phase fractions. Kinetic measurements of these fractions for both populations were demonstrated. CONCLUSIONS: The fluorochrome-to-protein ratio is important and can be optimized objectively for these assays. A permeabilization-sensitive antigen (p105), previously requiring formaldehyde/detergent-fixed cell preparations, was shown to work equally well with formaldehyde/ methanol fixation. Three-laser, two-parameter intracellular antigen analysis can be successfully coupled with DNA content analysis. Cell cycle kinetic analysis of endoreduplicating populations should be improved.
Benzimidazoles↗
Estimation of kinetic cell-cycle-related gene expression in G1 and G2 phases from immunofluorescence flow cytometry data.
BACKGROUND: Flow cytometry of immunofluorescence and DNA content provides measures of cell-cycle-related gene expression (protein and/or epitope levels) for asynchronously growing cells. From these data, time-related expression through S phase can be directly measured. However, for G1, G2, and M phases, this information is unavailable. We present an objective method to model G1 and G2 kinetic expression from an estimate of a minimum biological unit of positive immunofluorescence derived from the distribution of specific immunofluorescence of mitotic cells. METHODS: DU 145 cells were stained for DNA, cyclin B1, and a mitotic marker (p105) and analyzed by flow cytometry. The cyclin B1 immunofluorescence (B1) distribution of p105-positive cells was used to model the B1 distribution of G2 and G1 cells. The G1/S and S/G2 interface measurements were used to calculate expression in S phase and test the validity of the approach. RESULTS: B1 at S/G2 closely matched the earliest modeled estimate of B1 in G2. B1 increased linearly through G1 and S but exponentially through G2; mitotic levels were equivalent to the highest G2 levels. G1 modeling of B1 was less certain than that of G2 due to low levels of expression but demonstrated general feasibility. CONCLUSIONS: By this method, the upper and lower bounds of cyclin B1 expression could be estimated and kinetic expression through G1, G2, and M modeled. Together with direct measurements in S phase, expression of B1 throughout the entire cell cycle of DU 145 cells could be modeled. The method should be generally applicable given model-specific assumptions.
Cell Cycle↗
Differentiation and assessment of cell death.
Three documented cell death pathways, apoptosis, necrosis, and oncosis will be discussed. The end result of each pathway is cell death; however, the path by which death is achieved and the morphological and physiological traits of each may be strikingly distinct. Now that well characterized models have been established for particularly apoptosis, the induction pathway(s) has received much attention and the pathway pathology is beginning to be understood. Three model systems were investigated: APO-1/Fas, hypoxia, and oncosis. Cell death was induced, and during a time course sampling, a variety of methodologies, including DNA fragmentation by flow cytometry and gel electrophoresis, DNA staining, flow cytometric light scatter, transmission electron microscopy, anti-tubulin, Trypan blue, annexin V, and anti-APO2.7 were employed to monitor the cell death progress. The apoptotic pathway in the CD95-induced Jurkat cell model was further investigated using caspase inhibitor peptides and analyzed for APO2.7 antigen expression and DNA fragmentation by flow cytometry. Time course sampling characterized the cell death pathway and helped to differentiate the capabilities of the methods. The time to response and duration of the response were dependent upon cell type and method of induction. The CD95-induced Jurkat cell model showed a classical apoptotic response; however the MDA-MB-175-VII hypoxia model and the anti-5A9 induced oncosis model were not as clear. Each methodology shows advantages and disadvantages that allow the investigator to select several methods to identify, monitor, and enumerate cells with respect to cell death progression using time course studies.
Cell Death↗
APO2.7 defines a shared apoptotic-necrotic pathway in a breast tumor hypoxia model.
A breast tumor hypoxia model used to simulate conditions which may exist within an enlarging tumor was examined using documented methods for identifying mechanisms of cell death and compared to the mitochondrial membrane-specific APO2.7 antigen expression. Hypoxic conditions were induced by holding cell pellets of MDA-MB-175-VII breast carcinoma cells in tightly capped centrifuge tubes for up to 10 days. Cells were harvested at 1.5, 3, 4.5, 6, 12, 18, and 24 h, and each 24 h thereafter to 10 days. APO2.7 was monitored in unprocessed cells (no permeabilization prior to staining) for all time points and processed cells (permeabilized prior to staining) for only the first 24 h. Cell viability probes trypan blue and anti-tubulin antibody showed a rapid increase in staining over the first 24 h, as did the phosphatidylserine-specific annexin V and DNA fragmentation by flow cytometry (range of 60-81% positive staining). Light scatter changes indicative of cell death were also quite remarkable. APO2.7 staining never exceeded 42% of the cell pellet over the 10 days of testing compared to greater than 95% staining for all other methods tested. When APO2.7 antigen expression was examined with respect to depth in the cell pellet, it was apparent that cells deeper in the pellet expressed APO2.7 more rapidly; however, fewer cells stained and cells showed fewer apoptotic features on an ultrastructural level than cells at the cell media interface. The study indicates that the anti-APO2.7 antibody may be able to discern apoptotic and incomplete apoptotic cells from necrotic MDA-MB breast cancer cells, traversing a heterogeneous pathway to cell death induced by hypoxia.
Antibodies, Monoclonal↗
Impaired expression and function of signal-transducing zeta chains in peripheral T cells and natural killer cells in patients with prostate cancer.
Detection of functional, circulating T cells and NK cells may serve as a clinical test for the selection of individuals who can benefit from immunotherapy. Incidence of the T-cell receptor zeta (TCRdelta) chain within these populations appears to correlate with adequate effector cell function. In patients with advanced malignancy, the absence or reduced expression of delta chain has been documented. Flow cytometric analysis in the present study revealed a significant reduction in delta chain expression in peripheral blood lymphocytes (PBL) of 14 of 22 prostate cancer patients (P < 0.000001) as compared to normal donors, apparent in both T cells (CD3+, CD4+, CD8+), and NK (CD16+) cells. Compared to normal donor PBL, patient PBL cultured in the presence of CD3 and CD28, also demonstrated reduced expression of CD69 and/or CD25, and in some cases, failed to activate at all. Furthermore, evidence of cell proliferation in activation-stimulated patient PBL was muted: average PCNA positivity equaled 14%, a marked difference from what was observed in normal donors (P < 0.0002). In 8 of 16 samples of PBL, where delta expression was originally low, delta levels returned to the normal range after 48 hour culture in serum-free medium, suggesting that the loss of delta is reversible and may be caused by a tumor-derived substance. These data support the premise that monitoring the expression of delta in a cancer patient may provide a unique insight into the immune status and functionality of the individual, with the potential to redirect or augment therapies and ultimately alter prognosis.
Adult↗
Comparison of flow cytometric DNA content analysis in fresh and paraffin-embedded ovarian neoplasms: a prospective study.
DNA ploidy analysis was performed on both fresh and paraffin-embedded preparations from each of 54 malignant ovarian neoplasms. Aneuploidy was detected in both the fresh and the paraffin-embedded tissue in 19 out of 54 (35%) malignant cases. In addition, aneuploidy was detected exclusively in fresh tissue in seven of the malignant cases, and exclusively in paraffin-embedded tissue in one of the malignant cases, yielding a total of 27 out of 54 (50%) aneuploid cases. The correlation coefficient (r-value) for fresh and paraffin-embedded tissue ploidy analysis in the malignant specimens was 0.91. Although the frequency of recurrence was higher and overall survival lower in the malignant aneuploid specimens of both types, the combined analysis of DNA and survival rates indicated superior prognostic significance of fresh tissue. Of the seven patients in whose specimens aneuploidy was detected exclusively in fresh tissue, all died of recurrent disease during the follow-up period. Our finding indicates that data generated by flow cytometry analysis of formalin-fixed tissue should be interpreted with caution before the data can be used to draw clinical inferences.
Adult↗
Monitoring early cellular responses in apoptosis is aided by the mitochondrial membrane protein-specific monoclonal antibody APO2.7.
A recently described mitochondrial membrane protein-specific monoclonal antibody, APO2.7, was examined for monitoring early apoptotic responses in anti-CD95 (7C11)-induced Jurkat cells. Jurkat cells were harvested at 1.5, 3, 4.5, 6, 12, and 18 h after induction of apoptosis, and APO2.7 antibody monitored in unprocessed (no permeabilization agent used prior to staining) and processed (permeabilized prior to staining) cells. Light-scatter changes (decreased forward-scatter and increased side-scatter) by flow cytometry were observed after 3 h, and detection of cell permeability in unprocessed cells, as measured by light microscopic examination of Trypan blue-stained cells and flow cytometric detection of tubulin, showed little change until after 6 h. In addition, unprocessed cells stained with APO2.7 antibody showed little increase in staining until after 6 h following induction of apoptosis, when DNA fragmentation was demonstrated by flow cytometry and gel electrophoresis; however, processed cells stained with APO2.7 antibody showed significant increase in staining after 1.5 h. Detection, using annexin V and flow cytometry, of phospholipid membrane asymmetry from exposure of phosphatidylserine showed greater, apparent nonspecific staining in noninduced cells as compared to the other markers of apoptosis, but nearly paralleled the results of APO2.7 staining in processed cells from 3-18 h following CD95 induction of apoptosis. The data presented herein indicate that the mitochondrial membrane protein-specific antibody, APO2.7, is useful as a marker for the detection of apoptotic cells.
Annexin A5↗
Discrimination of late apoptotic/necrotic cells (type III) by flow cytometry in solid tumors.
A method is described for the discrimination of Type III, late apoptotic, and necrotic cells, to improve the accuracy of proliferation and ploidy determinations of breast tumors. We selected an immunological probe, antitubulin antibody, and a DNA specific stain, propidium iodide (PI), both capable of crossing the permeable membranes of Type III, late apoptotic, and necrotic cells. This study utilized MDA-MB-175-VII breast carcinoma cells deprived of oxygen for up to 11 d to simulate intratumoral hypoxia, and 10 human breast tumors and mouse-human breast tumor xenografts disassociated by mechanical or enzymatic means. After 24 h under hypoxic conditions, the MDA cells displayed characteristics associated with both apoptosis and necrosis. Approximately 50% of day 1 cells showed membrane permeability by trypan blue and absence of DNA laddering; however, by day 3-4 characteristic apoptotic DNA laddering by gel electrophoresis was evident. Substantial DNA content loss, further evidenced by a reduction in PI staining and fluorescent microscopy, was obvious by day 5. By day 10, 98% of cells showed no propidium iodide staining by conventional PI live/dead cell gating, but were positive for antitubulin antibody staining. When the study was extended to the analysis of ten tumors, antitubulin antibody showed a range of 78%-96% staining with a median value of 87.5%, while PI staining showed a range of 8%-74% with a median value of 11.5%. This study demonstrates that a large percentage of cells in tumors and hypoxic cell populations have significantly reduced DNA content, such that conventional live/dead cell gating using PI may include many Type III cells as live cells, thus significantly altering data involving multicolor investigations.
Animals↗
Lymphocytes subsets in normal individuals: analysis by four color immunofluorescence and flow cytometry on whole blood.
In order to precisely define human lymphocytes subsets, we used four color immunofluorescence analysis and flow cytometry. We report here the results of systematic studies performed on whole blood from 20 normal volunteers. This was performed using monoclonal antibodies recognizing 18 different CD molecules and analyzed here into 14 different 4-color immunofluorescence combinations. This showed that among T cells, the two major CD3+CD8+ and CD3+CD8- subsets differed greatly in the percentage of cells expressing CD26, CD27, CD28, CD38, CD45RA, CD45RO, CD57, S6F1, BY55, CD101 and their respective combination representing an array of subsets. Furthermore the minor NK subsets, i.e. CD2+CD3-CD8+, CD2-CD3-CD8+, CD2+CD3-CD8-, CD2-CD3-CD8- also differed in percentages of cells expressing CD16, CD56, CD57, S6F1, BY55 and their combinations.
Antigens, CD↗
Use of a multiparametric panel to target subpopulations in a heterogeneous solid tumor model for improved analytical accuracy.
The exclusion of non-tumor and dead cells from the analysis of live tumor cells can significantly improve the accuracy of prognostic indicators such as proliferative and DNA indexes. To target live breast tumor cells in a heterogeneous breast tumor model, we have designed a panel consisting of the DNA-specific dye DAPI and epithelial tissue-specific (cytokeratin), tumor-associated (MC5), proliferation-associated (proliferating cell nuclear antigen), and viability-associated (tubulin) markers. The breast tumor model consisted of a mixture of equal numbers of live and dead MDA-MB-175-VII (breast tumor) cells, live CEM (leukemic) cells, and live peripheral blood mononuclear cells. Targeting the live MDA cells in the mixture by gating on tubulin, cytokeratin, and MC5 resulted in a sevenfold increase in PCNA positivity (from 3% ungated to 22.3%), a 60% decrease in the %S-phase fraction (from 37.2% ungated to 15%), and elimination of extraneous hypodiploid and diploid components, enriching the tetraploid MDAs. These results are consistent with those obtained from unmixed live MDA cells. The combined utilization of this panel and "cumulative" electronic gating of the targeted population increases the number of relevant parameters that can be analyzed per sample and the accuracy of the resultant data.
Biomarkers, Tumor↗
Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry.
Dead cells represent a significant source of interference in the flow cytometric analysis of viable cells primarily due to nonspecific uptake of probes, increased autofluorescence, and altered antigen expression and DNA content. Traditional methods of dead cell exclusion, based on light scatter or uptake of dyes such as propidium iodide (PI) or fluorescein diacetate (FDA), are appropriate for the analysis of fresh, relatively homogeneous samples. However, they are incompatible with the development in this laboratory of a solid tumor monoclonal antibody panel incorporating combined surface and intracellular staining: Light scatter is unreliable in heterogeneous samples such as solid tumors, and most of the widely used viability probes are incompatible, due to weak or reversible binding, with the use of permeabilizing agents for intracellular staining. To determine the best viability marker for inclusion in the solid tumor panel, we compared cultured cells held under hypoxic conditions for up to 15 days after harvest, stained with eight viability probes, and processed according to the solid tumor panel procedure (unprocessed cells from each day, stained with PI, were used as standards). The viability probes included PI (in processed and unprocessed samples); 7-aminoactinomycin D (7-AAD); TO-PRO-3; laser dye styryl (LDS)-751; ethidium monoazide (EMA); and actin, cytokeratin, and tubulin indirectly labelled with sheep-alpha-mouse-FITC (SAM-FITC). The selection criteria for the best viability probe included broad cell type specificity: low nonspecific staining of live cells, specific staining of dead cells strong enough to withstand the permeabilization procedure, high signal-to-noise ratio throughout the time course, and compatibility with the four other fluorescent probes making up the tumor antibody panel. TO-PRO-3, LDS-751, and PI (in processed cells) stained both live and dead cells indiscriminately. Actin-SAM-FITC, EMA, and 7-AAD did not display sufficiently high signal-to-noise ratios over the entire time course. Cytokeratin-SAM-FITC was acceptable in every respect other than its specificity only for cells of epithelial origin. Tubulin-SAM-FITC alone satisfied all the criteria and was selected for inclusion in the monoclonal antibody panel as a viability probe.
Azides↗
Methodologies for the preservation of proliferation associated antigens PCNA, p120, and p105 in tumor cell lines for use in flow cytometry.
The retention of antigen expression of PCNA, p120, and p105 in two tumor cell lines (MOLT-4 and MDA-MD-175-VII) under various conditions of fixation was investigated using flow cytometric analysis. Four currently utilized procedures for fixation/permeabilization of intracellular antigens were compared for their ability to stain the nuclear antigens. A procedure using a brief incubation in a solution of lysolecithin in paraformaldehyde followed by fixation in ice-cold methanol prior to antibody staining was selected to evaluate reagent protocols aimed at preserving antigen expression. Holding samples overnight at 4 degrees C in 2.5% fetal bovine serum after the lysolecithin/paraformaldehyde and methanol fixation steps prior to staining with monoclonal antibodies resulted in no decline in the percentage of cells positively stained for all three markers with little decrease in intensity of fluorescence and no increase in DNA coefficient of variation (c.v.). Fixed/permeabilized MOLT-4 cells held longer than 24 h before staining were lower in PCNA fluorescence than freshly stained cells; holding samples of either cell line longer than 48 h resulted in decreased PCNA and p120 staining. Prefixing and holding cells in 50% methanol or 50% ethanol overnight before processing and staining severely depressed PCNA and p120 fluorescence. Prefixing either cell line in a range of concentrations (0.25-1.0%) of paraformaldehyde also resulted in reduced intensity of PCNA and p120 fluorescence along with increased DNA c.v. P105 staining appeared to be relatively unaffected by all prefixation/storage conditions tested, except for a decline of fluorescence when MDA cells were prefixed in 50% ethanol. Cells cryopreserved in liquid nitrogen for 1 week before processing showed < 5% loss of PCNA and p120 fluorescence compared to freshly processed cells, but p105 fluorescence dropped 29% in cryopreserved MDA cells. These results underscore the fact that specific protocols for the fixation and storage of biological samples prior to staining and analysis must be determined for the specific nuclear antigen marker under investigation.
Cell Cycle↗
Intratumor variability in prognostic indicators may be the cause of conflicting estimates of patient survival and response to therapy.
The DNA index, percentage of S-phase cells, proliferation fraction, and glutathione (GSH) content were determined at more than 1100 separate sites in 140 human tumors and 140 normal tissues. The study showed that the variability was so great from site to site within a tumor that there was only a 61% chance of identifying an aneuploid tumor clone (when present) if only a single site sample was analyzed for DNA content. Similar broad variability was observed in the percentage of S-phase cells, proliferation fraction, and glutathione content. Since these tumor characteristics are often used to predict the outcome of therapy and patient survival, the inaccuracy and underestimation of the test results may cause conflicting or erroneous predictions. The probability of finding an aneuploid clone or elevated percentage of S-phase cells proliferation fraction and GSH content increased dramatically as the number of sample sites studied per tumor was increased. Statistical analyses indicated that in order to achieve a 90% probability that the test results for these parameters were representative of the whole tumor: (a) all single site testing should be abandoned; (b) assays should be performed on samples taken from 3-7 different sites within each tumor; or (c) samples from each tumor should be pooled and the analyses run on a thoroughly mixed or homogenized aliquot of the multisite sample.
Breast Neoplasms↗
Expression of proliferation-associated antigens (PCNA, p120, p145) during the reentry of G0 cells into the cell cycle.
Flow cytometric bivariate analysis was used to evaluate the expression of PCNA, p120, and p145 during the G0 reentry of CHO-K1 cells into the cell cycle. CHO-K1 cells were placed in a G0-like state using serum depletion and stimulated to reenter the cell cycle by replating into fresh, serum-containing medium. At discrete intervals after stimulation, replicate samples were stained for either PCNA, p120, or 145; stained for DNA (Coulter DNA-Prep); evaluated on the EPICS Profile I; and analyzed on the EPICS ELITE workstation. PCNA stained less than 10% of the G0 cells; in contrast, however, 30-35% of the G0 cells were positive for p120 and p145. Eight hours after stimulating G0 cells to reenter the cell cycle (during G0/G1), p120 reached 88% positivity, while p145 and PCNA were 63% and 30% positive, respectively. Cells in S phase (12 and 16 h following G0 stimulation) were greater than 90% positive for all three antigens. PCNA had the greatest change throughout the G0 reentry process, both in percentage positive and quantitatively (mean channel fluorescence). This report indicates that all three proliferation-associated antigens studied are differentially expressed during the reentry of G0 cells into the cell cycle. Furthermore, these antigens may be useful in the early detection of G0 recruitment.
Animals↗