Basic Applied Biology

Basic Applied Biology

Temperature of liquid nitrogen used in cryopreservation is:

Liquid nitrogen at atmospheric pressure liquefies at minus 196 Celsius, providing ultra-low temperature environment where molecular diffusion essentially ceases, enzymatic catalysis halts, aqueous solutions enter amorphous glassy state rather than crystalline ice if cooling rapid enough, and cellular aging arrested for decades to centuries. This temperature lies well below glass transition of water-cryoprotectant mixtures around minus 130 Celsius where dangerous devitrification and ice recrystallization occur, ensuring stability during long-term storage. Freezers at minus 80 Celsius or minus 2

Ref: Mazur Journal General Physiology 1984 cryobiology principles; ATCC cryopreservation guide LN2 minus 196; Alberts cell storage methods.

View Details

Basic Applied Biology

Cryoprotectant commonly used for animal cells is:

Cryopreservation enables essentially indefinite storage of mammalian cell lines and primary cells by arresting biochemical reactions at cryogenic temperatures, but prevention of lethal intracellular ice formation crucial. Dimethyl sulfoxide at 5 to 10 percent volume per volume emerged as standard cryoprotectant for most animal cells due to rapid membrane permeability coefficient 10^-5 cm per second, colligative depression of freezing point, ability to replace water molecules via hydrogen bonding and promotion of vitrification rather than crystallization that would mechanically puncture organel

Ref: Pegg Principles of Cryopreservation 2015; Freshney Ch freezing and cryopreservation; PubMed cryoprotectant toxicity PMID 21846505.

View Details

Basic Applied Biology

Cells are passaged optimally at confluency of:

Subculturing or passaging maintains exponential growth, prevents phenotypic drift associated with contact inhibition, nutrient exhaustion, waste accumulation and extracellular matrix overproduction. For adherent animal cells optimal confluency for passage ranges 70 to 80 percent, stage where monolayer covers majority of flask surface but retains intercellular gaps, active mitosis indicated by refractile rounded mitotic figures, high viability exceeding 95 percent via trypan blue exclusion, and low expression of cyclin-dependent kinase inhibitors p27KIP1 and p21 that accumulate in quiescence. A

Ref: Freshney Ch subculture guidelines; Alberts Ch cell cycle contact inhibition mechanisms; ATCC passaging standard operating procedures.

View Details

Basic Applied Biology

Phenol red in culture media acts as:

Phenol red chemically phenolsulfonphthalein, sulfonephthalein class dye, serves as visual pH indicator deliberately incorporated at 5 to 15 mg per L in many mammalian culture media including Dulbecco Modified Eagle Medium, RPMI 1640 and Minimum Essential Medium to allow non-invasive monitoring of acid-base status. Color transition results from protonation equilibrium with pKa approximately 7.9 at 37 Celsius and ionic strength of media: acidic protonated form absorbs at 430 nanometer appearing yellow below pH 6.8, partially deprotonated neutral form appears orange-red around pH 7.4 physiologica

Ref: Freshney Ch indicators; Berthois et al. Proc Natl Acad Sci USA 1986 phenol red estrogen; NCBI media formulation guide.

View Details

Basic Applied Biology

Optimum CO₂ concentration for RPMI medium is:

RPMI 1640 medium formulated by George Moore and colleagues at Roswell Park Memorial Institute in 1966 specifically for suspension culture of human leukocytes and hematopoietic cells, exhibits unique formulation low in calcium 100 mg per L and magnesium reduced to minimize aggregation, enriched with vitamins and reduced glutathione. Crucially it relies on sodium bicarbonate buffer system 2.0 g per L interacting with gas phase carbon dioxide via equilibrium CO2 plus H2O equilibrates to H2CO3 equilibrates to H+ plus HCO3- described by Henderson-Hasselbalch equation. Five percent CO2 atmosphere pr

Ref: Moore et al. JAMA 1967 RPMI development; Freshney Ch media and supplements; ATCC cell culture guide RPMI buffer.

View Details

Basic Applied Biology

Primary cell culture is defined as:

Primary cell culture defines culture establishment directly from living tissue obtained via biopsy, surgical resection, embryonic dissection or enzymatic disaggregation without prior in vitro passage or immortalization step, preserving in vivo characteristics. Tissue fragments minced to one cubic millimeter pieces, digested with collagenase type I, trypsin-EDTA, dispase or liberase cocktails to release single cells, filtered to remove debris and seeded into tissue culture flasks containing medium supplemented with serum providing growth factors, antibiotics preventing contamination and attachm

Ref: Freshney Culture of Animal Cells 7th ed. Ch 11 Primary culture; Alberts Molecular Biology of Cell Ch cell culture methods.

View Details

Basic Applied Biology

Immunotoxins kill target cells by:

Immunotoxins constitute chimeric fusion therapeutics coupling targeting domain, typically single-chain variable fragment scFv or Fab derived from monoclonal antibody recognizing tumor-associated antigen like CD22 or HER2, to potent catalytic toxin of bacterial or plant origin including Pseudomonas exotoxin A fragment PE38, diphtheria toxin DT390, ricin A chain, saporin or gelonin. Mechanism begins with antigen-specific binding, internalization via clathrin-mediated endocytosis into early endosome, pH-dependent furin cleavage between targeting and toxin domains, retrograde transport via KDEL re

Ref: Pastan et al. Nature Reviews Cancer 2006 Immunotoxins; Weldon & Pastan FEBS J 2011 mechanism; PubMed PMID 16794637.

View Details

Basic Applied Biology

Rituximab targets antigen:

Rituximab represents first chimeric anti-cancer monoclonal antibody approved 1997 targeting CD20, a 33 to 37 kDa non-glycosylated tetra-span membrane phosphoprotein encoded by MS4A1 gene on chromosome 11q12 expressed on pre-B through mature B lymphocytes but absent on hematopoietic stem cells, pro-B cells and terminally differentiated plasma cells. Protein architecture comprises four transmembrane domains with short intracellular termini and two extracellular loops accessible for antibody binding. Physiologic function involves regulation of calcium flux through modulating B cell receptor signa

Ref: Maloney et al. Blood 1997 Rituximab ideal target; Reff et al. CD20 biology; PubMed review CD20 therapeutic target.

View Details

Basic Applied Biology

HAT medium selects hybridoma cells based on:

HAT medium selection developed by John Littlefield in 1964 exploits differential nucleotide anabolism pathways to isolate hybrid cells from mixture of parental populations. De novo purine and pyrimidine synthesis requires tetrahydrofolate-dependent enzymes, specifically aminopterin, a folate analog, competitively inhibits dihydrofolate reductase DHFR blocking de novo pathway. Alternative salvage pathway recycles hypoxanthine via hypoxanthine-guanine phosphoribosyltransferase HGPRT converting to IMP and thymidine via thymidine kinase TK to TMP, bypassing blocked de novo routes. Myeloma partners

Ref: Littlefield Science 1964 Selection of hybrids; Abbas Immunology 10th ed. HAT selection; Alberts Ch nucleotide metabolism salvage.

View Details

Basic Applied Biology

Hybridoma technology produces:

Hybridoma technology developed by Georges Köhler and César Milstein in 1975 and awarded Nobel 1984 created paradigm shift by immortalizing antibody-producing B lymphocytes through somatic cell fusion. Mice immunized with antigen develop germinal center reaction where B cells undergo VDJ recombination, somatic hypermutation and class switching to generate high affinity plasma cells. Splenic lymphocytes harvested shortly after boost are fused with HGPRT-deficient myeloma partner such as SP2/0-Ag14 or NS0 using polyethylene glycol 1500 that perturbs lipid bilayers promoting membrane fusion formin

Ref: Köhler & Milstein Nature 1975 Continuous cultures of fused cells; Abbas Cellular and Molecular Immunology 10th ed. monoclonal antibody generation.

View Details

Basic Applied Biology

Electrochemical biosensors mainly detect changes in:

Electrochemical biosensors dominate commercial diagnostic market, typified by blood glucose strips, due to ease of miniaturization, low cost and compatibility with portable potentiostats. In these devices analyte recognition by oxidoreductase, dehydrogenase, or affinity bioreceptor immobilized on working electrode modifies local redox environment, ion concentration or interfacial electron transfer resistance. Amperometric mode measures current arising from oxidation or reduction of enzymatic product such as hydrogen peroxide at fixed potential following Cottrell equation where current proporti

Ref: Wang Chemical Reviews 2008 Electrochemical Biosensors; Lodish Molecular Cell Biology 9th ed. biosensing; NIH biosensor guide.

View Details

Basic Applied Biology

Biosensor component converting biological signal to electrical signal is:

Biosensor architecture integrates three functional modules working in concert: bioreceptor such as enzyme, antibody, aptamer or whole cell conferring molecular recognition specificity, transducer that converts biorecognition event into quantifiable physical signal, and signal processing display unit amplifying and presenting output. Transducer role remains pivotal because biochemical interactions like enzyme-substrate turnover producing protons or hydrogen peroxide, antibody-antigen binding causing mass change, or DNA hybridization changing charge do not inherently generate electrical signals

Ref: Tuan Vo-Dinh et al. Biosensors and Bioelectronics Handbook; Alberts Cell Ch signal transduction devices; Nature Biotech 2020 review.

View Details