Flow cytometry, FACS, ELISA

Flow cytometry, FACS, ELISA

Final cell count per well after 27-fold dilution of 2.7×10⁶ cells/ml and seeding 100 μl is:

Cell counting for seeding uses dilution calculation C1V1 equals C2V2. Starting concentration 2.7 times ten to six cells per ml divided 27 fold yields 1 times ten to five cells per ml. Seeding 100 microliters equals 0.1 ml per well. Multiplying concentration by volume gives cells per well: 1 times ten to five times 0.1 equals 1 times ten to four. Earlier stated 2.7 times ten to four would be intermediate before final volume correction. Final count accounts for both dilution factor and aliquot volume ensuring accurate plating density.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details

Flow cytometry, FACS, ELISA

Which ELISA format is best for measuring hormone levels like insulin?

Competitive ELISA is preferred for low molecular weight analytes present at low concentrations such as hormones insulin, triiodothyronine and steroids where sandwich assay fails due to single epitope and small size preventing dual antibody binding. Sample hormone competes with enzyme labeled hormone for limited antibody binding sites coated on plate. Signal inversely proportional to hormone level allows sensitive quantification in nanomolar range with small sample volume. Direct and indirect formats lack required sensitivity, sandwich requires two non overlapping epitopes unavailable on small hormones, making competitive format ideal.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details

Flow cytometry, FACS, ELISA

Direct ELISA detects:

Direct ELISA is simplest format where antigen from sample is adsorbed directly to polystyrene plate through passive hydrophobic interactions. After blocking, enzyme conjugated primary antibody specific to antigen is added, binding directly without secondary antibody. Substrate addition generates signal directly proportional to antigen present. It detects antigen not antibody, unlike indirect ELISA which detects antibodies. Only one antibody layer reduces steps but lowers amplification and specificity compared to sandwich formats. Its utility lies in measuring purified antigens or screening antibody specificity with enzyme labeled primary antibody.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details

Flow cytometry, FACS, ELISA

What happens to radioactive signal in RIA if antigen is high?

Radioimmunoassay principle involves fixed limited antibody, fixed trace amount of radiolabeled antigen, and variable unlabeled antigen from sample competing for binding. At equilibrium, higher concentration of unlabeled antigen occupies more antibody sites, displacing labeled antigen into free fraction that is washed away. Radioactivity measured in bound pellet therefore decreases as sample antigen increases. This inverse relationship allows construction of standard curve. No change or direct proportionality would violate competition kinetics, equal distribution would indicate no affinity. Bound counts fall with rising antigen levels.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details

Flow cytometry, FACS, ELISA

What is the use of flow cytometry?

Flow cytometry analyzes physical and chemical properties of individual cells suspended in fluid. Hydrodynamic focusing forces single file passage through laser intercept, measuring forward scatter proportional to size, side scatter indicating granularity, and fluorescence from labeled antibodies or dyes reflecting antigen expression, DNA content or calcium flux. It does not sequence DNA, assess protein tertiary folding or amplify nucleic acids like PCR. Applications include immunophenotyping, cell cycle analysis, apoptosis detection, sorting of specific populations for culture, making it powerful for quantitative cellular property measurement.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details

Flow cytometry, FACS, ELISA

Luciferin used with luciferase results in:

Luciferase enzyme from fireflies and marine organisms catalyzes oxidation of substrate luciferin in ATP and oxygen dependent reaction. Energy released does not produce color change but photon emission in visible range, termed bioluminescence. No external excitation light is needed, unlike fluorescence of GFP which requires light source. Reaction produces low background and high sensitivity detection used in reporter assays, ATP quantification and immunoassays. Green fluorescence would require fluorophore excitation, while luciferin luciferase system generates intrinsic light production measurable by luminometer for quantitative biology.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details

Flow cytometry, FACS, ELISA

Which is detected in competitive ELISA?

Competitive ELISA is advantageous for small molecules, haptens and subtle structural differences that cannot accommodate two antibodies simultaneously in sandwich format. One antigenic site allows competition between labeled reference and sample antigen for limited antibody. Minor variations in epitope structure alter competitive displacement curves, enabling discrimination of analogs, drug metabolites, hormone isoforms and pesticide residues. Antibody subclass identification needs isotype specific secondaries, protein folding requires biophysical methods, cell counting needs cytometry. Sensitive competition reveals small antigenic differences undetectable by direct binding alone.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details

Flow cytometry, FACS, ELISA

Which tag can increase chemiluminescence in ELISA?

Colorimetric ELISA uses HRP, ALP and beta galactosidase to produce colored products, but sensitivity limited. Chemiluminescence enhances detection using enzymes or proteins generating light in presence of substrate. Luciferase catalyzes luciferin oxidation producing photons detectable by luminometer, vastly amplified compared to absorbance. GFP is fluorescent not chemiluminescent, ALP can also produce chemiluminescence with appropriate dioxetane substrates but luciferase is archetypal bioluminescent tag increasing signal to noise ratio. Fusion of luciferase to antibody improves limit of detection for low abundance antigens.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details

Flow cytometry, FACS, ELISA

Which assay is best to detect apoptotic cells?

Viability assays measure different aspects of cell health. MTT measures metabolic activity of mitochondria, trypan blue assesses membrane permeability, chromium release measures cytotoxic lysis. None specifically identify programmed cell death early stage. Annexin V FITC flow cytometry detects externalized phosphatidylserine, earliest membrane event in apoptosis while membrane remains impermeable to PI, allowing distinction of early and late apoptosis versus necrosis. Quantitative fluorescence per cell and population percentages make it sensitive, specific and standard for apoptosis kinetics in drug screening and immunology research laboratories.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details

Flow cytometry, FACS, ELISA

In flow cytometry, light is converted from analog to digital in which step?

Flow cytometry signal processing follows sequence: cells generate light via scatter and fluorescence, photodetectors and photomultipliers collect photons and convert into analog voltage pulses proportional to intensity. Preamplifiers amplify these weak signals. Analog to digital converters digitize pulse height, area and width into numerical values stored for analysis. Amplification occurs before conversion to preserve dynamic range. Thus analog to digital conversion is third major step after light generation and analog amplification, enabling computerized gating, compensation and statistical analysis of thousands of events per second.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details

Flow cytometry, FACS, ELISA

In RIA, diabetic patients show:

Radioimmunoassay is competitive binding assay where fixed amount of radiolabeled antigen competes with unlabeled patient antigen for limited antibody. Bound radioactivity is inversely proportional to patient antigen concentration. In type one diabetes low insulin secretion occurs due to beta cell destruction. With little cold insulin to compete, antibody binds predominantly hot labeled insulin, so precipitated complex retains high radioactivity counts. High insulin sample would displace hot insulin and lower counts. Therefore diabetic insulin deficient samples show low circulating insulin but high bound radioactivity reading, indicating hormone deficiency.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details

Flow cytometry, FACS, ELISA

O-Phenyl diamine is used as a chromogenic substrate in?

ELISA relies on enzyme conjugated antibodies converting chromogenic substrates into colored products measurable spectrophotometrically. Horseradish peroxidase commonly uses TMB, ABTS and O phenylenediamine OPD producing orange brown soluble product read at 492 nm after acid stop. Alkaline phosphatase uses pNPP. OPD offers good sensitivity and stability for peroxidase detection. RIA uses radiolabeled isotopes measured by gamma counter, Southern and Western blotting may use chemiluminescence or colorimetric substrates but OPD is classic ELISA chromogen, not used in nucleic acid hybridization or radioactivity detection protocols.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details