Flow Cytometric Analysis of Fluorescent Estrogen Binding in Cancer Cell Suspensions
Louis P. Pertschuk, Sin Hang Lee in Localization of Putative Steroid Receptors, 2019
Flow cytometry (FCM) began over 25 years ago as a tool to automatically count and size individual cells from a flowing cell suspension. By the mid 1950s, this early tool was commercially available as the Coulter Counter®. Ten years later, with the aid of more sophisticated spectrophotometry detection and computer coupling, multiparameter analysis of DNA or protein staining cellular constituents could be performed on cells flowing at the rate of 500/sec. These developments resulted in the later availability of the Ortho Cytofluorograph®. Fluorescent dye emission rather than absorption was utilized to increase the signal-to-noise ratio, and the demonstration of stoichiometric binding between fluorochromes and DNA expanded the application of FCM to producing DNA histograms and analyzing the growth of tumor cell suspensions for percent G1, S, and G2/M phase cells. Most recently, the Becton-Dickinson Fluorescence Activated Cell Sorter (FACS)® introduced electrostatic sorting of individual cells based on the analysis of light scatter (cell size) or fluorescence intensity. The application of this new and expanding field to medical and biologic problems has been extensively reviewed by Melamed et al.14 It is now clear that FCM can be successfully used to study the binding of a wide variety of fluorescently labeled ligands to their membrane, cytoplasmic, or nuclear receptors in intact cells.15
Alternative Methodologies to Animal Testing
Nicola Loprieno in Alternative Methodologies for the Safety Evaluation of Chemicals in the Cosmetic Industry, 2019
Two assays for testing in vitro teratogens have been used by the National Toxicology Program (NTP) in the U.S.: The MOT assay measures the attachment of ascitic mouse ovarian tumor cells (MOT cells) to a lectin (concanavalin A-) coated surface. Cellular adhesiveness is a generic property of embryonic cells; therefore, it is assumed that inhibition of adhesiveness by a chemical indicates teratogenic potential. The test chemical is added to a suspension of MOT cells which have been exposed to radiolabeled thymidine, and a plastic sheet coated with concanavalin A is placed in the culture vessel. Over a period of hours, the MOT cells normally adhere to the lectin-coated surface. Inhibition of this phenomenon by teratogens can be assessed by counting the radioactivity associated with the plastic sheets and by comparison with control values.The HEMP assay measures the growth and proliferation of Human Embryonic Palatal Mesenchyme (HEMP) cells in the presence of test agents. Growth and division of cells is a fundamental process in developing tissues, and inhibition of this would be indicative of developmental toxicity. The HEMP cells are plated at a low density in tissue culture dishes. After 24 h, the test agent is added, and the cultures are maintained for an additional 72 h without a media change. At the end of this period, the number of cells present is counted using a Coulter counter.
The HbS Containing Cell
Ronald L. Nagel in Genetically Abnormal Red Cells, 2019
The average level of Hb in sickle cell anemia patients is sex and age dependent. According to the data of Serjeant et al.175 and Hayes et al.,176 the hemoglobin level is significantly lower than normal controls as early as 2 weeks after birth and reaches a plateau at 3 to 6 months, with a further decrease ending at the 15th month. The sex dependence is rather complex, with lower Hb levels in males before puberty and a dramatic reversal after that period which is attributable to the steep increase in Hb levels observed among males between the ages of 10 and 20. At age 20 the average among females is slightly under 8 g/dℓ and about 8.5 g/dℓ among males, according to the Jamaican sample. Red cell indices from Coulter counters are generally normal, not reflecting the enormous range in MCV and MCHC detected by other methods (reviewed previously in this chapter). A combination of cancelling differences and intrinsic Coulter counter artifacts accounts for this discrepancy. When MCHC data is obtained using the centrifuged microhematocrit, the data are less affected by artifact but still reflect only averages. With this type of MCHC data the Jamaican authors have detected some small differences between SS and AA MCHCs in children that might well be related to differences in iron deficiency between the two groups.175 Coexisting alpha thalassemia have an effect on average red cell indices when the effect of the reticulocytosis is taken into account.106 As discussed previously in the section on red cell heterogeneity, isopycnic gradients are a more powerful tool to study and detect these changes.
Prevalence, risk factors and outcome of Mycoplasma pneumoniae infection among children in Uganda: a prospective study
Published in Paediatrics and International Child Health, 2021
Rebecca Nantanda, Freddie Bwanga, Irene Najjingo, Grace Ndeezi, James K Tumwine
The blood for IgM serological tests was immediately transferred into a cold box at 4°C. The IgM used was Demeditec Mycoplasma pneumoniae IgM ELISA DEMYCM0350 (Demeditec Diagnostics GmbH, Germany). The sample for CBC was kept at room temperature until transferral to the laboratory. All samples reached the laboratory within 8 hours of collection. The Coulter counter method was used to measure the total and differential white cell count. The sputum quality was assessed both macroscopically to determine whether it was saliva and by the Gram stain method. Gram stain quantified the number of organisms in the sputum, and all samples with >10 squamous epithelial cells per low power field were considered to be of poor quality. The PCR results from such samples were not analysed for M. pneumoniae. In these participants, only the results of the IgM for M. pneumoniae were considered.
Measuring the Systemic Inflammatory Response to On- and Off-Pump Coronary Artery Bypass Graft (CABG) Surgeries Using the Tryptophan/Kynurenine Pathway
Published in Journal of Investigative Surgery, 2022
Ahmed Farouk, Rasha A. Hamed, Saeid Elsawy, Nashwa F. Abd El Hafez, Farag M. Moftah, Muammar A. Y. Nassar, Fify Alfy Gabra, Tahia H. Saleem
From each patient, 5 mL of venous blood was withdrawn according to the schedule: 24 h preoperative, immediately after complete revascularization, 10 min before the end of operation and 72 h postoperative. About 3 mL of each sample was collected on heparin tube for Trp and Kyn estimation while 2 mL was collected in plain collection tubes for IL-6 determination. WBCs were analyzed by coulter counter as a routine investigation. Serum and plasma were separated by centrifugation at 3500 rpm for 15 min at 4 °C, and then stored in −20 °C freezer. Plasma acidic protein was precipitated by sulfosalicylic acid and centrifugation, for determination of Trp. The free amino acids remaining in the supernatant were used for analysis by HPLC using ion exchange column.
3D high resolution clonogenic survival measurement of xrs-5 cells in low-dose region of carbon ion plans
Published in International Journal of Radiation Biology, 2023
Dea Kartini, Olga Sokol, Chutima Talabnin, Chinorat Kobdaj, Marco Durante, Michael Krämer, Martina Fuss
For the monolayer samples, approximately 2 5 cells were seeded in 25 cm2 culture flasks and incubated for 24 h before irradiation. To recover cells from the monolayer, medium was removed and cells were rinsed with PBS (phosphate buffered saline, Gibco). Next, 1 mL of trypsin (0.25% Trypsin − 0.02% EDTA in Hank’s Balanced Salt Solution, Sigma) was added, and cells were incubated for 5 min until detaching from the surface. Subsequently, 3 mL of medium was added to deactivate the trypsin and the cell suspension was collected. Cells were counted with a Beckman Coulter counter using a profile with 7 − 18 µm particle size.
Related Knowledge Centers
- Blood Smear
- Hematology
- Microfluidics
- Microscope
- Flow Cytometry
- Electrolyte
- Resistive Pulse Sensing
- Solution
- Medical Laboratory
- Complete Blood Count