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Why would you plate cells at different densities in a colormetric assay?

Why would you plate cells at different densities in a colormetric assay?


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The aim of my experiment is to see if a kinase inhibitor reduces cancer cell viability. I am using 2 different cell densities 50,000 cells/well and 100,000 cells/well and different doses of the kinase inhibitor.


The comments you have given are some reasons, although they also depend on the confluency of the cells. Your teacher/professor/advisor is also making sure that you can see an effect at low levels of drug, as well as making sure that changes in the signal are visible - for example, does a 50% reduction in viability due to drug activity give the same drop in signal as reducing the number of cells by 50%? You don't say what kind of assay you're running, but a fairly common and inexpensive one is the MTT assay, with some kits nowadays switching to XTT for greater sensitivity and a reduction in the number of steps in the assay (example kit from Cell Signaling compared to an MTT assay kit from ThermoFisher), as the final colorimetric product does not require solubilization before reading. Finally, depending on the lab you're in, the conditions for the assay may not have been optimized yet, so your instructor wants you to test a couple of different densities to see what works best.


Useful Numbers for Cell Culture

There are various sizes of dishes and flasks used for cell culture. Some useful numbers such as surface area and volumes of dissociation solutions are given below for various size culture vessels.

Catalog No.Surface area (cm 2 )Seeding density*Cells at confluency*Versene
(mL of 0.05% EDTA). Approx. volume
Trypsin
(mL of 0.05% trypsin, 0.53 mM EDTA). Approx. volume
Growth medium
(mL). Approx. volume
Dishes
35mm1504601503188.80.3 x 10 6 1.2 x 10 6 112
60mm15046215028821.50.8 x 10 6 3.2 x 10 6 335
100mm15046415035056.72.2 x 10 6 8.8 x 10 6 5512
150mm1504681683811455.0 x 10 6 20.0 x 10 6 101030
Culture plates
6-well1406759.60.3 x 10 6 1.2 x 10 6 111 to 3
12-well1506283.50.1 x 10 6 0.5 x 10 6 0.4 to 10.4 to 11 to 2
24-well1424751.90.05 x 10 6 0.24 x 10 6 0.2 to 0.30.2 to 0.30.5 to 1.0
48-well1506871.10.03 x 10 6 0.12 x 10 6 0.1 to 0.20.1 to 0.20.2 to 0.4
96-well1670080.320.01 10 6 0.04 x 10 6 0.05 to 0.10.05 to 0.10.1 to 0.2
Flasks
T-25156367156340250.7 x 10 6 2.8 x 10 6 333–5
T-75156499156472752.1 x 10 6 8.4 x 10 6 558–15
T-1751599101599201754.9 x 10 6 23.3 x 10 6 171735–53
T-2251599341599332256.3 x 10 6 30 x 10 6 222245–68
*Seeding density is given for each culture vessel type as follows: Dishes and Flasks: Cells per vessel Culture plates: Cells per well.

†The number of cells on a confluent plate, dish, or flask will vary with cell type. For this table, HeLa cells were used.


Assay Protocol to Measure Cytotoxicity

Additional Reagents Required:

  • Culture medium, e.g., RPMI 1640 (R0883) containing 10% heat inactivated FCS (fetal calf serum, 12106C), 2 mM glutamine (G6392) and 1 μg/ml actinomycin C1 (actinomycin D, A9415).
  • If an antibiotic is to be used, additionally supplement media with penicillin/streptomycin or gentamicin
  • Tumor necrosis factor-α, human (hTNF-α) (10 μg/ml), sterile (T6674).

Protocol:

For the determination of the cytotoxic effect of human tumor necrosis factor-α (hTNF-α, T6674) on WEHI-164 cells (mouse fibrosarcoma, 87022501) (Figure 2).

  1. Preincubate WEHI-164 cells at a concentration of 1 × 10 6 cells/ml in culture medium with 1 μg/ ml actinomycin C1 for 3 h at 37 °C and 5-6.5% CO2.
  2. Seed cells at a concentration of 5 × 10 4 cells/ well in 100 μl culture medium containing 1 μg/ml actinomycin C1 and various amounts of hTNF-α (final concentration e.g., 0.001–0.5 ng/ml) into microplates (tissue culture grade, 96 wells, flat bottom).
  3. Incubate cell cultures for 24 h at +37 °C and 5-6.5% CO2.
  4. After the incubation period, add 10 μl of the MTT labeling reagent (final concentration 0.5 mg/ml) to each well.
  5. Incubate the microplate for 4 h in a humidified atmosphere (e.g., +37 °C, 5-6.5% CO2).
  6. Add 100 μl of the Solubilization solution into each well.
  7. Allow the plate to stand overnight in the incubator in a humidified atmosphere (e.g., +37 °C, 5-6.5% CO2).
  8. Check for complete solubilization of the purple formazan crystals and measure the absorbance of the samples using a microplate (ELISA) reader. The wavelength to measure absorbance of the formazan product is between 550 and 600 nm according to the filters available for the ELISA reader, used. The reference wavelength should be more than 650 nm.

Figure 2. Determination of the cytotoxic activity of recombinant human TNF-α (hTNF-α) on WEHI-164 cells (mouse fibrosarcoma) using MTT assay.


Overview

Product name

Detection method

Sample type

Assay type

Assay time

Product overview

Extracellular Oxygen Consumption Assay Kit ab197243 is a mix-and-read, 96-well fluorescence plate reader assay for the real-time kinetic analysis of extracellular oxygen consumption rates (OCR). The oxygen consumption rate is a measure of the cellular respiration rate, and of mitochondrial function.

The assay is optimized for isolated mitochondria and cell cultures, and can be used with tissues, enzyme preparations, and small organisms.

The fluorescent dye used in this assay kit is quenched by oxygen. The dye excites at 360-380 nm (max 380) and emits at 630-680 nm (max 650). It is also available separately as ab197242.

In the assay, an oil layer is added on top of the assay medium to limit diffusion of oxygen into the assay medium. As mitochondrial respiration depletes the oxygen within the assay medium, quenching of the fluorescent dye is reduced, and the fluorescence signal increases proportionately.

The reaction is non-destructive and fully reversible (the oxygen sensitive dye is not consumed) enabling assay time courses and drug treatments.

Notes

Or review the full metabolism assay guide for other assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress.


Isopycnic Centrifugation in Non-Ionic Media

Factors Affecting the Analysis of Gradients

As stated previously, saccharidic solutes inhibit the activity of enzymes, and some enzymes are more sensitive than others. Therefore, when studying the distribution of marker enzymes across a gradient the sucrose concentrations in each assay should be adjusted so that they are similar. Sucrose also interferes with the estimation of glycogen, the orcinol estimation of RNA, the diphenylamine estimation of DNA, the micro-biuret estimation of protein and decreases the sensitivity of the Lowry protein assay procedure ( Hinton, Burge and Hartman, 1969 Hartman et al., 1974 ). However, monosaccharides and disaccharides can easily be removed by, for example, acid-precipitation of the sample, or pelleting the material after dilution, or dialysis.

When measuring the radioactivity of gradient fractions, the addition of water-soluble scintillant can give rise to precipitation of the sugar solute. However, such problems can be avoided by diluting samples or by careful choice of the scintillant (see p. 54 ). Even in the absence of precipitation, the sucrose in the samples can quench β-emitters, particularly the weak β-emission of 3 H-labelled compounds ( Dobrota and Hinton, 1973 ).


Clonogenic assay of cells in vitro

Clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. The colony is defined to consist of at least 50 cells. The assay essentially tests every cell in the population for its ability to undergo “unlimited” division. Clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing radiation, but can also be used to determine the effectiveness of other cytotoxic agents. Only a fraction of seeded cells retains the capacity to produce colonies. Before or after treatment, cells are seeded out in appropriate dilutions to form colonies in 1–3 weeks. Colonies are fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v) and counted using a stereomicroscope. A method for the analysis of radiation dose–survival curves is included.


Results & Discussion

Fig. 2 shows the percentage reduction of alamarBlue ® after being averaged and including the standard deviations. Greater alamarBlue ® reduction (i.e. higher levels of cell growth) is observed for substrate A over the whole culture period. In both substrates, cell proliferation increased with culture time over the first 15 days. The metabolic activity of the cells growing on both substrates seems to slow-down by day 15, suggesting that the surfaces were advancing into confluence.

Fig. 2: Percentage reduction of alamarBlue as a function of culture time, for substrates A and B.

RAPID METHODS FOR FOOD HYGIENE INSPECTION

Matthias Upmann , Christine Bonaparte , in Encyclopedia of Food Microbiology , 1999

Optical Methods

Colorimetry and fluorometry: Specific physical or chemical changes (pH, oxidation/reduction potential, enzymatic transformations) associated with microbial metabolic activity can be indicated by changes in colour, fluorescence or colour intensity of an added reagent dye during sample incubation. Many chromogenic and fluorogenic dyes are used depending on the metabolic change to be shown. A multitude of miniaturized and computer-aided or even fully automated identification systems for pure cultures are based on this principle.

Colorimetry and fluorometry can also be used for quantitative purposes by measuring the required incubation time in order to produce a colour reaction or fluorochrome formation. Broadly known indicators are litmus and bromocresol purple for detecting pH shifts or resazurin, methylene blue, and triphenyltetrazolium chloride as oxidation/reduction indicators.

Fully automated procedures are now available which use reflectance colorimeters or fluorometers. These techniques are applicable for rapid estimation of total microbial numbers or specific microorganisms, for product shelf-life stability, starter culture activity, and antibiotic testing. Fur further details see Table 2 .

Turbidimetry: Increasing cell numbers lead to an increase in optical density of liquid growth media. Therefore, a light beam will increasingly we weakened on transillumination when a liquid sample is incubated. By varying the sample dilution, the growth medium and the incubation temperature the result can be narrowed to specific bacterial species or numbers. Some methodological properties are given in Table 2 .

Turbidimetry is widely applied in vitamin bioassays and disinfectant testing. It has been used for sterility testing in food quality control. Its application may be limited by background turbidity of foods (fat globules, blood cells, food particles).

Pyruvate determination: Pyruvate is a key compound in bacterial lactose metabolism and can serve as an indicator for milk quality monitoring. Pyruvate is measured indirectly by spectrophotometric detection of reduced nicotinamide–adenine dinucleotide (NADH) which is a cofactor in the enzymatic breakdown of pyruvate. Since somatic cells contribute to the pyruvate content of milk and not all bacteria produce pyruvate, the relation between this metabolite and total microbial count is limited (see Table 2 ).


Similarities Between MTT and MTS Assay

  • MTT and MTS assay are two types of assays that determine cell viability in vitro.
  • Both assays help to assess the effect of test molecules on cell proliferation and cytotoxicity.
  • Also, both are colorimetric assays.
  • Furthermore, they assess the metabolic activity of cells based on the capability of cells to reduce the tetrazolium dye to its formazan.
  • In addition, the enzyme responsible for the above reduction is NAD(P)H-dependent cellular oxidoreductase, which is present in viable cells.
  • Moreover, the reduction reaction occurs outside the cell by plasma membrane electron transport.
  • The MTT reagent is light-sensitive hence, these assays have to be performed in dark.
  • Besides, the incubation time for both assays is the same after the addition of tetrazolium dye and it is 1 to 4 hours at 37°C.

A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing

The incidence of fungal infections has increased significantly over the past decades. Very often these infections are associated with biofilm formation on implanted biomaterials and/or host surfaces. This has important clinical implications, as fungal biofilms display properties that are dramatically different from planktonic (free-living) populations, including increased resistance to antifungal agents. Here we describe a rapid and highly reproducible 96-well microtiter-based method for the formation of fungal biofilms, which is easily adaptable for antifungal susceptibility testing. This model is based on the ability of metabolically active sessile cells to reduce a tetrazolium salt (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide) to water-soluble orange formazan compounds, the intensity of which can then be determined using a microtiter-plate reader. The entire procedure takes approximately 2 d to complete. This technique simplifies biofilm formation and quantification, making it more reliable and comparable among different laboratories, a necessary step toward the standardization of antifungal susceptibility testing of biofilms.


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