Mineralization assay

steps to create a standard curve (6)

prepare a series of standard solutions w/ known [] of the analyte; measure the response (eg fluorescence) of each standard solution using an appropriate instrument, ensure you measure the response of the blank (0 []) as well; plot the data - plot the measured response (y-axis) against the known [] (x axis) on a graph - create scatterplot; fit a curve to the data points (often linear is appropriate); determine the equation of fitted line/curve - will be used to calculate the [] of unknown sample; calculate unknowns - measure response of unknown samples and use equation of standard curve to calculate the [] of unknown samples

what is alizarin red used for

to visually detect the presence of mineralization in bone tissue

why do you wash cells first in alizarin red staining?

to remove residual culture medium. Medium contains salts, proteins, and serum that can react with the dye, causing high background noise and non-specific staining that obscures your calcium deposits

what do you do after washing alizarin red

fix cells with formaldehyde

why do you rinse cells 3 times after removing formaldehyde alizarin red

preventing chemical interference with the dye and avoiding background staining

why do you add water to alizarin red staining at end just before visual inspection/image aquisition?

to prevent cells from drying

why do you adjust the pH to 4.1 alizarin red?

to ensure the dye specifically binds to calcium deposits; prevents false positives

what is ALP

alkaline phosphatase - Hydrolase enzyme responsible for removing phosphate groups from many types of molecules, most effective in an alkaline environment, sometimes used synonymously as basic phosphatase.

- in assay breaks down substrate into yellow-colored nitrophenol or phosphate

what does ALP say about whether or not a cell is an osteoblast?

if it contains it - it may be an osteoblast
if it doesnt contain it - definitely not an osteoblast

what is a MgCl/AMP buffer for ALP assay

allows for pH stabilization; AMP does transphosphorylation (enzyme transfers the phosphate group to the AMP molecule), the transphosphorylation speeds up the enzyme reaction - stronger and more accurate signal; MgCl2 acts as an enzymatic cofactor,

what is the stop solution for ALP assay

NaOH - strong base - stops all enzyme action

function of p-nitrophenyl phosphate ALP assay

chromogenic substrate - acts as the specific target for the ALP enzyme, which catalyzes the dephosphorylation of pNPP 

what is the dilution reagens in a calcium assay

acetic acid

chelator meaning

chemical compound that binds tightly to metal ions, immobilizing them in a ring-like structure

ethanolamine/boric acid solution function in calcium assay

ensures accurate and vivid color development, ethanoalamine acts as the primary alkalizing agent, boric acid acts as a buffering agent to stabilize pH of the reagent, second addition of ethanolamine is to completely dissolve the boric acid

components of working solution calcium assay

solution A - ethanolamine/boric acid buffer
solution B - OCPC (o- cresophtalein complexone, metallochromic indicator and colorimetric chromogen)
solution C - hydroxyquinoline (orange) - magnesium masking agent, prevents magnesium ions reacting w/ calcium dye

what do you need for a DNA assay

lysis buffer - to liberate DNA

ALP/DNA assay descr

DNA measurement used to normalize ALP activity, so you can accurately compare enzyme levels across samples w/ diff cell numbers - total ALP activity divided by total DNA mass, ensuring data reflects cell-specific activity rather than just total cell mass

Cyquant cell proliferation steps (5)

grow cells in microplate wells, remove culture media, freeze, thaw and add dye in lysis buffer, measure fluoresence

ALP what color means what

measure absorbance at 405nm, the more yellow the more enzymatic activity

what is CQ

green fluorescent dye that exibits strong fluorescence when bound to cellular nucleic acids