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Fulltext Lipoprotein-induced cell growth and hemocyanin biosynthesis in rhogocytes

Sairi F, Gomes VG, Dehghani F, Valtchev P

Cell Tissue Res, 2022 May;388(2):359-371.
PMID: 35088179 DOI: 10.1007/s00441-022-03577-1

Rhogocyte is a unique molluscan cell that synthesises a supramolecular respiratory protein known as hemocyanin. Its ability to synthesise the protein has eluded the scientists despite hemocyanin's importance as a carrier protein and complex molecule with anti-viral activity. Although a hypothetical model of hemocyanin release from the rhogocytes lacunae was proposed based on colloid-osmotic pressure mechanism, lack of in vitro studies limits further validation of this model. In this study, we aim to investigate the impact of cell culture conditions and nature of hemocyanin biosynthesis of rhogocyte cells dissociated from Haliotis laevigata mantle tissue. Population of cells with different hemocyanin expression levels was profiled using flow cytometry, while hemocyanin concentrations in the media were elucidated by ELISA assay. We demonstrated that addition of lipoprotein supplement into the media resulted in a burst secretion of hemocyanin into the culture media. Over 7 days of culture, the population of cells tagged with hemocyanin antibody increased steadily while hemocyanin release in the media decreased significantly. Variation of culture medium, temperature, growth supplement type and concentration also impacted the cell growth and hemocyanin biosynthesis. These results indicated the possibility of an active process triggered by the addition of supplement to synthesise the protein at the highest amount during the first hour. The current study provides a glimpse of the hemocyanin biosynthesis by rhogocyte that may be significant to understand the cell ability to synthesise supramolecular protein and secretion through lacunae.

Matched MeSH terms: Flow Cytometry
Fulltext Single Cell Electrical Characterization Techniques

Mansor MA, Ahmad MR

Int J Mol Sci, 2015;16(6):12686-712.
PMID: 26053399 DOI: 10.3390/ijms160612686

Electrical properties of living cells have been proven to play significant roles in understanding of various biological activities including disease progression both at the cellular and molecular levels. Since two decades ago, many researchers have developed tools to analyze the cell's electrical states especially in single cell analysis (SCA). In depth analysis and more fully described activities of cell differentiation and cancer can only be accomplished with single cell analysis. This growing interest was supported by the emergence of various microfluidic techniques to fulfill high precisions screening, reduced equipment cost and low analysis time for characterization of the single cell's electrical properties, as compared to classical bulky technique. This paper presents a historical review of single cell electrical properties analysis development from classical techniques to recent advances in microfluidic techniques. Technical details of the different microfluidic techniques are highlighted, and the advantages and limitations of various microfluidic devices are discussed.

Matched MeSH terms: Flow Cytometry/methods*
Quantitative Flow Cytometry-Based Assays for Measuring Constitutive Secretion

Gordon DE, Shun-Shion AS, Asnawi AW, Peden AA

Methods Mol Biol, 2021;2233:115-129.
PMID: 33222131 DOI: 10.1007/978-1-0716-1044-2_8

Constitutive secretion is predominantly measured by collecting the media from cells and performing plate-based assays. This approach is particularly sensitive to changes in cell number, and a significant amount of effort has to be spent to overcome this. We have developed a panel of quantitative flow cytometry-based assays and reporter cell lines that can be used to measure constitutive secretion. These assays are insensitive to changes in cell number making them very robust and well suited to functional genomic and chemical screens. Here, we outline the key steps involved in generating and using these assays for studying constitutive secretion.

Matched MeSH terms: Flow Cytometry/methods*
Fulltext Comparison between flowcytometry and immunoperoxidase staining for the enumeration of lymphocyte subsets

Dhaliwal JS, Malar B, Quck CK, Sukumaran KD, Hassan K

Singapore Med J, 1991 Jun;32(3):163-5.
PMID: 1876889

Immunoperoxidase staining was compared with flowcytometry for the enumeration of lymphocyte subsets. The percentages obtained for peripheral blood lymphocytes using immunoperoxidase (CD3 = 76 CD4 = 27.9, B = 10.7 CD4/CD8 = 1.8) differed significantly from those obtained by flowcytometry (CD3 = 65.7 CD4 = 39.4, CD8 = 25.6, B = 16.7, HLA DR = 11.9 CD4/CD8 = 1.54) for certain subsets (CD3, CD4, B). There was no significant difference in lymphocyte subsets between children and adults using the same method. These differences are probably due to the different methods used to prepare lymphocytes for analysis. Other factors that should also be considered are the presence of CD4 antigen on monocytes and CD8 on natural killer cells.

Matched MeSH terms: Flow Cytometry/methods*
Early T-cell precursor lymphoblastic leukaemia with monocytic morphology negative for CD3 by flow cytometry: A diagnostic challenge solved by immunohistochemistry

Hsieh YC, Wu HC, Chuang SS

Malays J Pathol, 2023 Aug;45(2):297-298.
PMID: 37658540

No abstract available.

Matched MeSH terms: Flow Cytometry
Detection of Circulating Tumor Cells and Epithelial Progenitor Cells: A Comprehensive Study

Fuloria S, Subramaniyan V, Gupta G, Sekar M, Meenakshi DU, Sathasivam K, et al.

J Environ Pathol Toxicol Oncol, 2023;42(3):1-29.
PMID: 37017676 DOI: 10.1615/JEnvironPatholToxicolOncol.2022044456

Technological advancement to enhance tumor cells (TC) has allowed discovery of various cellular bio-markers: cancer stem cells (CSC), circulating tumor cells (CTC), and endothelial progenitor cells (EPC). These are responsible for resistance, metastasis, and premetastatic conditions of cancer. Detection of CSC, CTC, and EPC assists in early diagnosis, recurrence prediction, and treatment efficacy. This review describes various methods to detect TC subpopulations such as in vivo assays (sphere-forming, serial dilution, and serial transplantation), in vitro assays (colony-forming cells, microsphere, side-population, surface antigen staining, aldehyde dehydrogenase activity, and Paul Karl Horan label-retaining cells, surface markers, nonenriched and enriched detection), reporter systems, and other analytical methods (flow cytometry, fluorescence microscopy/spectroscopy, etc.). The detailed information on methods to detect CSC, CTC, and EPC in this review will assist investigators in successful prognosis, diagnosis, and cancer treatment with greater ease.

Matched MeSH terms: Flow Cytometry
Comparative analysis of three permeabilization methods for cytofluorometric evaluation of cytoplasmic myeloperoxidase

Tay SP, Cheong SK, Hamidah NH, Ainoon O

Malays J Pathol, 1999 Jun;21(1):37-43.
PMID: 10879277

A comparative study was conducted to evaluate three different permeabilization methods: FACS Permeabilizing Solution (FPerm), CytoFix/CytoPerm Kit (CFP) and Paraformaldehyde-Tween 20 (PFT) reagents, in cytoplasmic labeling of myeloperoxidase (MPO). Peripheral blood cells from 23 healthy subjects were fixed and permeabilized according to the proposed procedures, prior to direct immunofluorescence staining with CD14, CD45, IgG1, IgG2 and MPO monoclonal antibodies (McAb). Subsequent flow cytometric analysis was performed on FACSCalibur flow cytometer (Becton Dickinson, BD). As far as the antigenic expression of MPO in normal samples is concerned, FPerm and CFP demonstrated better cytoplasmic staining by inducing minor effects on light-scattering properties of the cell populations, whereas PFT-treated samples showed a diminished ability to distinguish the cell types. However, the simple and rapid FPerm method required an earlier processing of samples since the stored whole blood samples (for more than 8 hours) tended to show a significant decrease of fluorescence intensity. We also have demonstrated that P/N ratio possesses added value in evaluation of cell reactivity in immunophenotyping, based upon the apparent nonspecific cytoplasmic staining of MPO in the lymphocyte population.

Matched MeSH terms: Flow Cytometry
Evaluating in-house anti-serum against B cells with flow cytometry

Chin SF, Cheong SK, Lim YC, Mok KL, Hamidah HN

Malays J Pathol, 1993 Dec;15(2):125-30.
PMID: 8065173

The applications of antibodies, be it monoclonal or polyclonal, in the diagnostic and research fields are well established. The disadvantage is the high cost of commercially available antibodies. In a diagnostic establishment like ours which also functions as a training ground for laboratory related personnel, it is beneficial to be able to produce in-house reagents. Therefore, we have undertaken this project to produce a rabbit polyclonal antibody against B lymphocytes. We found that the rabbit was a good choice because the titre of antibody produced was high and positive reactions were still detected at a dilution of 1:38400. The antibody showed significant positive reaction only with the lymphocyte subpopulation. A positive reaction was observed between the immunized rabbit serum and B lymphocytes but not T lymphocytes. This shows that the antibody was B lymphocyte specific. There was a positive correlation between the percentage of B lymphocytes labelled using the commercial anti-CD19 monoclonal antibody and the in-house polyclonal antibody (n = 13, r = 0.7, p = 0.02). However, the percentage of cells labelled by the in-house polyclonal anti-B was lower than that by the commercial monoclonal anti-CD19. The fluorescence intensity of the polyclonal antibody was lower than that of the monoclonal. In general, the performance of the in-house polyclonal antibody can be considered as satisfactory. The rabbit serum was stored at -20 degrees C and no significant loss of activity was detected for over a period of 19 months.

Matched MeSH terms: Flow Cytometry
Fulltext CD89/CD35 Expression Ratio in Salivary Neutrophil as an Early Detection Marker for Severe Early Childhood Caries

Luthfi M, Oki AS, Indrawati R, Rifai M, Dachlan YP, Razak FA

Eur J Dent, 2020 Jul;14(3):386-392.
PMID: 32645730 DOI: 10.1055/s-0040-1713704

OBJECTIVES: To analyze CD35/CD89 expression ratio on the surface of neutrophils as an early detection marker for S-ECC.
MATERIALS AND METHODS: Saliva was collected from 4- to 6-year-old kindergarten students. Salivary neutrophils were obtained by instructing the subjects to rinse their mouth with 1 mL of sterile 1.5% NaCl for 30 seconds before expectorating it into a sterile glass. The expression of CFSE+CD35+ and CFSE+CD89+was measured and analyzed using flow cytometry.
RESULTS: The expression of CFSE+CD89+ in the caries-free group (2.46 ± 0.39) was significantly lower than that in the S-ECC group (3.41 ± 1.11), with a p-value of 0.0001, while the expression of CFSE+CD35+ in the caries-free group was (2.35 ± 0.56) compared with (1.54 ± 0.35) (p = 0.0001) in the S-ECC group.
CONCLUSIONS: The expression ratio of CFSE+CD89+ and CFSE+CD35+constitutes a marker for S-ECC.

Matched MeSH terms: Flow Cytometry
Fulltext The estimation of foetomaternal haemorrhage by flowcytometry

Nurasyikin, Y., Suria, A.A., Ng, Soon Peng, Leong, C.F.

Medicine & Health, 2015;10(2):112-122.
MyJurnal

Fetomaternal haemorrhage (FMH) may occur following a sensitizing event, during pregnancy or at delivery. In cases of rhesus (Rh) incompatibility between mother and the fetus, it can thus subject to the haemolytic disease of the newborn. The Kleihauer test for quantification of FMH lacks standardization and results are less accurate. Furthermore, it cannot differentiate the foetal cell from the adult HbF. Flowcytometry analysis using monoclonal antibodies, is a new technique for the quantification of FMH and it allows larger number of cells to be analysed. It is also able to differentiate the foetal cell from maternal HbF, and thus is more sensitive and accurate. The objective of our study was to determine the FMH using the flowcytometric analysis of anti-HbF antibody and to correlate the FMH using flow cytometry and the standard Kleihauer test. Ninety eight peripheral blood samples from pregnant women at more than 20 weeks of pregnancy and post delivery were analyzed by both methods. The percentage of the foetal cells were recorded and the FMH were calculated. We found a fair correlation between the two methods with the correlation coefficient r = 0.633 (p

Matched MeSH terms: Flow Cytometry
Fulltext Quantification of intracellular payload release from polymersome nanoparticles

Scarpa E, Bailey JL, Janeczek AA, Stumpf PS, Johnston AH, Oreffo RO, et al.

Sci Rep, 2016 07 11;6:29460.
PMID: 27404770 DOI: 10.1038/srep29460

Polymersome nanoparticles (PMs) are attractive candidates for spatio-temporal controlled delivery of therapeutic agents. Although many studies have addressed cellular uptake of solid nanoparticles, there is very little data available on intracellular release of molecules encapsulated in membranous carriers, such as polymersomes. Here, we addressed this by developing a quantitative assay based on the hydrophilic dye, fluorescein. Fluorescein was encapsulated stably in PMs of mean diameter 85 nm, with minimal leakage after sustained dialysis. No fluorescence was detectable from fluorescein PMs, indicating quenching. Following incubation of L929 cells with fluorescein PMs, there was a gradual increase in intracellular fluorescence, indicating PM disruption and cytosolic release of fluorescein. By combining absorbance measurements with flow cytometry, we quantified the real-time intracellular release of a fluorescein at a single-cell resolution. We found that 173 ± 38 polymersomes released their payload per cell, with significant heterogeneity in uptake, despite controlled synchronisation of cell cycle. This novel method for quantification of the release of compounds from nanoparticles provides fundamental information on cellular uptake of nanoparticle-encapsulated compounds. It also illustrates the stochastic nature of population distribution in homogeneous cell populations, a factor that must be taken into account in clinical use of this technology.

Matched MeSH terms: Flow Cytometry
Fulltext Effect of fetal bovine serum on erythropoietin receptor expression and viability of breast cancer cells

Teo GY, Rasedee A, Al-Haj NA, Beh CY, How CW, Rahman HS, et al.

Saudi J Biol Sci, 2020 Feb;27(2):653-658.
PMID: 32210684 DOI: 10.1016/j.sjbs.2019.11.032

Erythropoietin receptors (EPORs) are present not only in erythrocyte precursors but also in non-hematopoietic cells including cancer cells. In this study, we determined the effect of fetal bovine serum (FBS) in culture medium on the EPOR expression and viability of the estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells. Using flow cytometry, we showed that the inclusion of 10% FBS in the medium increased the EPOR expressions and viabilities of MDA-MB-231 and MCF-7 cells. The MDA-MB-231 showed greater EPOR expression than MCF-7 cells, suggesting that the presence of ERs on cells is associated with poor expression of EPOR. Culture medium containing 10% FBS also caused increased number of breast cancer cells entering the synthesis phase of the cell cycle. The study also showed that rHuEPO treatment did not affect viability of breast cancer cells. In conclusion, it was shown that the inclusion of FBS in culture medium increased expression of EPOR in breast cancer cells and rHuEPO treatment had no effect on the proliferation of these cancer cells.

Matched MeSH terms: Flow Cytometry
Fulltext Inhibitory Activity of Ficus deltoidea var. trengganuensis Aqueous Extract on Lipopolysaccharide-Induced TNF-α Production from Microglia

Wang H, Vidyadaran S, Mohd Moklas MA, Baharuldin MTH

Evid Based Complement Alternat Med, 2017;2017:2623163.
PMID: 29358962 DOI: 10.1155/2017/2623163

Objective: To explore the effect of Ficus deltoidea (FD) aqueous extracts on the release of tumor necrosis factor-α (TNF-α), the expression of CD40, and the morphology of microglial cells in lipopolysaccharide- (LPS-) activated BV2 cells.
Methods: The cytotoxicity of FD extract was assessed by MTS solution. BV2 cells were divided into 5 experimental groups, intervened, respectively, by FD (4 mg/mL) and LPS + FD (0, 1, 2, and 4 mg/mL). Besides, a blank control group was set up without any intervention. TNF-α release was assessed by enzyme linked immunosorbent assay (ELISA). The expression of CD40 was examined by flow cytometry. Immunocytochemical staining was used to show the morphology of BV2 cells.
Results: FD extract of different concentrations (1, 2, and 4 mg/mL) had no significant toxic effects on the BV2 cells. FD suppressed the activation of microglia in morphology and reduced TNF-α production and expression of CD40 induced by LPS.
Conclusion: FD extract has a therapeutic potential against neuroinflammatory diseases.

Matched MeSH terms: Flow Cytometry
Fulltext Qualitative flow cytometric analysis of Malaysian myelodysplastic syndromes (MDS) patients

Mohadese Hashem B, Ramasamy R, Sabariah MN, Seman Z

Med J Malaysia, 2012 Feb;67(1):77-80.
PMID: 22582553 MyJurnal

Myelodysplastic syndromes (MDS) are a group of haematological malignancies categorized by ineffective hematopoiesis that result in dysplasia. Although morphological diagnosis is a traditional and standard technique that is used for the diagnosis of MDS, the heterogeneous blood and bone marrow characteristics of MDS patients can potentially obscure the right diagnosis. Thus, we have utilized flow cytometric immunophenotyping as a supportive mechanism to obtain a more accurate and faster method for detection of abnormal markers in MDS. Flow cytometry was used for analyzing bone marrow samples from newly diagnosed MDS patients to investigate the abnormal antigen expression patterns in granulocytic, monocytic, erythroid, lymphoid lineages and myeloid precursors. The results were compared with those obtained from cases that had Idiopathic Thrombocytopenic Purpura (ITP) as a control. The most common abnormality found in the granulocytic lineage was the decrease of CD10. Low expressions of CD13 were the most frequent abnormality in the monocytic lineage. The erythroid lineage was found to have low expression of CD235A+/CD71+, reduce of CD71 and decreased CD235a. In conclusion, this method is useful for confirming cases in which it is difficult to make a diagnosis by morphology.

Matched MeSH terms: Flow Cytometry/methods*
Intracellular antigen determination by flow cytometry in leukaemic cells

Chin SF, Cheong SK

Malays J Pathol, 1994 Jun;16(1):69-73.
PMID: 16329579

Several fixation and permeabilization techniques that enable the flow cytometric analysis of the cell contents have been introduced in recent years. These methods allow sensitive detection of intracellular antigens that facilitates the diagnosis of certain diseases. We have undertaken in this study to evaluate a simple method of fixation and permeabilization using 2% paraformaldehyde and Tween 20. Intracellular antigens in three different leukaemia cases were analysed. We found that the method was reliable and easy. Intracellular kappa light chains were found in abundance in a case of plasma cell leukaemia. CD3 and CD22 were found in greater amount intracellularly than on the surface in pre-T-ALL and pre-pre B-ALL respectively.

Matched MeSH terms: Flow Cytometry/methods*
Isolation and characterization of primary microglia from post-natal murine brain tissues: a comparison of two methods

Jose S, Tan SW, Tong CK, Vidyadaran S

Cell Biol Int, 2015 Dec;39(12):1355-63.
PMID: 26194799 DOI: 10.1002/cbin.10516

Microglia are resident macrophages of the central nervous system (CNS). Apart from playing vital roles as sentinel cells, they are crucial in physiological processes such as synaptic pruning during brain development. CNS disorders require an understanding of the contribution of each cellular compartment to the pathogenesis. Elucidating the role of microglia in disease development and progression in the intricate CNS environment is technically challenging and requires the establishment of reliable, reproducible techniques to isolate and culture microglia. A number of different protocols have been developed for isolation of neonatal microglia and here we compare two widely used methods, namely, mild trypsinization and EasySep® magnetic separation. EasySep® magnetic separation provided higher microglia yield, and flow cytometric evaluation of CD11b and F4/80 markers revealed that EasySep® separation method also produced significantly higher purity compared to mild trypsinization. Microglia isolated using EasySep® separation method were functional, as demonstrated by the generation of nitric oxide, IL-6, TNF-α, and MCP-1 in response to lipopolysaccharide stimulation. In summary, this study has revealed that magnetic separation is superior to mild trypsinization in terms of yield and purity of microglia.

Matched MeSH terms: Flow Cytometry/methods*
Fulltext Measuring pH of the Plasmodium falciparum digestive vacuole by flow cytometry

Abu Bakar N

Trop Biomed, 2015 Sep;32(3):485-93.
PMID: 26695209 MyJurnal

Studies show that the pH of the malaria parasite's digestive vacuole (DV) plays a key role in the physiological functions of this organelle and antimalarial drug accumulation, and yet is technically difficult to measure. In this study, a flow cytometry-based technique was developed to measure the DV pH using a ratiometric pH indicator, FITC-dextran loaded into the DV of saponin-permeabilized parasites. To calculate the DV pH, a standard pH calibration curve was generated by incubating the saponin-permeabilized cells in buffers with different pH in the presence of an ionophore, CCCP. The measured average pH of the DV was 5.27 ± 0.03 that is approximately the same in the parasites observed microscopically by Hayward et al. (2006) (5.50 ± 0.14) using the same probe. The removal of glucose from the medium, causing a rapid depletion of parasite ATP, resulted in an alkalization of the DV. The DV was reacidified upon restoration of glucose to the medium. This technique provides a rapid, simple and quantitative measurement of the DV pH on a large number of cells. It will also be useful in future attempts to evaluate the effect of antimalarial drugs (i.e. chloroquine and artemisinin-based drugs) in pH changes of the DV.

Matched MeSH terms: Flow Cytometry/methods*
Immunophenotyping analysis of lymph node biopsies by flow cytometry

Raja-Sabudin RZ, Hamid AA, Yusof N, Alauddin H, Aziz SA, Kulaveerasingam S, et al.

Saudi Med J, 2012 Oct;33(10):1131-3.
PMID: 23047221
Matched MeSH terms: Flow Cytometry*
Fulltext Antigen expression pattern of acute promyelocytic leukaemia cases in Malaysia

Ambayya A, Zainina S, Salmiah MS, Sabariah MN

Med J Malaysia, 2014 Apr;69(2):64-9.
PMID: 25241814 MyJurnal

INTRODUCTION: Acute Promyelocytic Leukaemia (APL) is associated with devastating coagulopathy and life threatening condition which requires immediate medical attention. It is crucial to establish an expedited diagnosis as early therapeutic intervention has led to optimal patient management. In this study, we assessed the type and frequency of antigen expressions in APL and correlated these findings with genetic studies.

METHODS: Multiparametric immunophenotyping was performed on 30 samples and findings were correlated with karyotypes, FISH for t(15;17) translocation and RT-PCR for PML-RARΑ for detection of breakpoint cluster regions (bcr1,bcr2 and bcr3).

RESULTS: On SSC/CD45, APL cells displayed high to moderate SSC, with the expression of CD33 (100%), CD13 (96.8%), cMPO (71%) but lacked CD34 (3.2%) and HLA-DR (9.7%). Aberrant expression of CD4 was seen in 12.9% and CD56 in 6.5% of the cases. A significant association between cumulative aberrant antigen expression and bcr1 were observed bcr1 (X2(2) =6.833,p.05) and (X2(2)=4.599,p>.05) respectively.

CONCLUSIONS: Flow cytometry is a rapid and effective tool in detecting APL. It is interesting to note that there is significant association between cumulative aberrant antigen expression and genotype analysis. Further validation is required to corroborate this relationship.

Matched MeSH terms: Flow Cytometry
Mesenchymal stromal cell-like characteristics of corneal keratocytes

Choong PF, Mok PL, Cheong SK, Then KY

Cytotherapy, 2007;9(3):252-8.
PMID: 17464757

The unique potential of mesenchymal stromal cells (MSC) has generated much research interest recently, particularly in exploring the regenerative nature of these cells. Previously, MSC were thought to be found only in the BM. However, further studies have shown that MSC can also be isolated from umbilical cord blood, adipose tissue and amniotic fluid. In this study, we explored the possibility of MSC residing in the cornea.

Matched MeSH terms: Flow Cytometry

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