A comparative study of colorimetric cell proliferation assays in immune cells

Abstract

Cell proliferation assays are basic and essential techniques for assessing cellular function. Various colorimetric assays, such as MTT-, WST-1-, and resazurin-based assays, are available; however, studies directly comparing the suitability of each method for immune cell proliferation are scarce. Thus, we aimed to determine the best reagent and its optimal conditions based on variables such as cell number range, stimulation dose, kinetics, and compatibility with the cell division assay using CFSE fluorescence dye which is able to directly monitor divided cells by flow cytometry. In the absence of stimulation, MTT solubilized with SDS (MTT-SDS) and resazurin appeared to accurately reflect the cell numbers in a linear fashion. On the other hand, WST-1 exhibited a higher stimulation index following strong stimulation, whereas MTT-SDS and resazurin exhibited a better sensitivity to weak stimulation. A longer duration for stimulation did not necessarily increase sensitivity. CFSE staining revealed incremental cell division in response to anti-CD3 antibody stimulation in a dose-dependent manner. The cell numbers indirectly estimated from cell division profiles were consistent with the dose-response curve in the absorbance of MTT-SDS and resazurin. The absorbance does not increase before cell division, irrespective of T cell activation status, suggesting that these reagents reflect the cell number but not the cellular volume. Collectively, resazurin and MTT-SDS seem to be more reliable than others, and thus appear applicable in various conditions for the immune cell experiments.

The Ability of Precursory Monocytes (MO) to Differentiate Varies Among Individuals But Is Stable Over Time

“…Use of the autogeneic system (both DCs and T cells are from the same individual) is dependent on time-related variability, and the MLR culture needs additional stimulus. Usually, vaccine antigen is used, but in our study we did not know the vaccination status of our donors [ 20 , 21 ].…”

“…In order to elicit this response, we used PMA and ION, in contrast to the bacterial pathogens used by others. PMA and ION are independent of the expression of PARP (poly[ADP-ribose] polymerase) receptors on the surface by direct activation of calcium influx and protein kinase C. Finally, we used MLR, but, predictably, we were able to establish that the level of T cell proliferation depends on major histocompatibility mismatch between IL-4 & GM-CSF differentiated MO and T cells [ 20 , 21 ].…”

Glutathione–Allylsulfur Conjugates as Mesenchymal Stem Cells Stimulating Agents for Potential Applications in Tissue Repair

“…Cell studies were conducted on cMCS and NHDF (Lonza, Basel, Switzerland) cell lines. cMSC were extracted by auricular biopsies made during the course of coronary artery bypass surgery from patients after signing a written consent form as previously described [38][39][40]89,90]. Cell cultures were grown in DMEM (Dulbecco's modified Eagle medium) (Gibco, Life Technologies, Milan, Italy), containing 10% v/v FBS (Fetal Bovine Serum) (Gibco, Life Technologies, Milan, Italy), 1% w/v penicillin-streptomycin (Sigma-Aldrich, Italy), and 1% w/v L-glutamine (Sigma-Aldrich, Italy).…”

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“…The intensity of developed color was measured by micro plate reader (Molecular device, USA) operating at 570 nm wavelength. 55 Percent cell viability was calculated by using the formula: (T/C) × 100, where C = Absorbance of control, T = Absorbance of treatment. The IC-50 values were calculated using the Origin software.…”