Crystal Violet Staining is a Reliable Alternative to Bicinchoninic Acid Assay-Based Normalization

Nilles, Julie; Weiß, Johanna; Theile, Dirk

doi:10.2144/btn-2022-0064

Cited by 14 publications

(10 citation statements)

References 12 publications

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“…To test this hypothesis, we designed ADCs that are susceptible or refractory to MUC16/CA125 HIO factor binding. Assays comparing their cytotoxicity against isogenic cell lines that were MUC16/CA125 positive or negative were quantified using methods confirmed to accurately measure cell growth kinetics and viability [ 23 ]. Our studies found that HIO factors can negatively impact alternative antibody-based formats such as ADCs similar to their parent IgG (Figs 2A and 3B ).…”

Section: Resultsmentioning

confidence: 99%

NAV-001, a high-efficacy antibody-drug conjugate targeting mesothelin with improved delivery of a potent payload by counteracting MUC16/CA125 inhibitory effects

2023

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Subsets of tumor-produced cell surface and secreted proteins can bind to IgG1 type antibodies and suppress their immune-effector activities. As they affect antibody and complement-mediated immunity, we call these proteins humoral immuno-oncology (HIO) factors. Antibody-drug conjugates (ADCs) use antibody targeting to bind cell surface antigens, internalize into the cell, then kill target cells upon liberation of the cytotoxic payload. Binding of the ADC antibody component by a HIO factor may potentially hamper ADC efficacy due to reduced internalization. To determine the potential effects of HIO factor ADC suppression, we evaluated the efficacy of a HIO-refractory, mesothelin-directed ADC (NAV-001) and a HIO-bound, mesothelin-directed ADC (SS1). The HIO factor MUC16/CA125 binding to SS1 ADC was shown to have a negative effect on internalization and tumor cell killing. The MUC16/CA125 refractory NAV-001 ADC was shown to have robust killing of MUC16/CA125 expressing and non-expressing tumor cells in vitro and in vivo at single, sub-mg/kg dosing. Moreover, NAV-001-PNU, which contains the PNU-159682 topoisomerase II inhibitor, demonstrated good stability in vitro and in vivo as well as robust bystander activity of resident cells while maintaining a tolerable safety profile in vivo. Single-dose NAV-001-PNU demonstrated robust tumor regression of a number of patient-derived xenografts from different tumor types regardless of MUC16/CA125 expression. These findings suggest that identification of HIO-refractory antibodies to be used in ADC format may improve therapeutic efficacy as observed by NAV-001 and warrants NAV-001-PNU’s advancement to human clinical trials as a monotherapy to treat mesothelin-positive cancers.

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Section: Resultsmentioning

confidence: 99%

NAV-001, a high-efficacy antibody-drug conjugate targeting mesothelin with improved delivery of a potent payload by counteracting MUC16/CA125 inhibitory effects

2023

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“…Antiproliferative effects of the rifamycins were investigated for all studied incubation periods using proliferation inhibition assays with crystal violet staining as described earlier (Nilles et al 2022a , b ; Peters et al 2006 ). Experiments were performed in three independent biological replicates with n = 8 wells for each concentration.…”

Section: Methodsmentioning

confidence: 99%

“…This was achieved by multiplying the measured mean cellular volume of a LS180 cell (CASY cell counter, Schärfe System, Reutlingen, Germany) by the mean cell number of four wells of an identically prepared and treated cell culture plate. Experiments were conducted in duplicate (one plate for uptake experiments and a second plate for normalization via crystal violet staining (Nilles et al 2022a , b )) with four biological replicates for each concentration.…”

Section: Methodsmentioning

confidence: 99%

Comprehensive in vitro analysis evaluating the variable drug–drug interaction risk of rifampicin compared to rifabutin

et al. 2023

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Compared to rifampicin (600 mg/day), standard doses of rifabutin (300 mg/day) have a lower risk of drug–drug interactions due to induction of cytochrome P450 3A4 (CYP3A4) or P-glycoprotein (Pgp/ABCB1) mediated by the pregnane X receptor (PXR). However, clinical comparisons with equal rifamycin doses or in vitro experiments respecting actual intracellular concentrations are lacking. Thus, the genuine pharmacological differences and the potential molecular mechanisms of the discordant perpetrator effects are unknown. Consequently, the cellular uptake kinetics (mass spectrometry), PXR activation (luciferase reporter gene assays), and impact on CYP3A4 and Pgp/ABCB1 expression and activity (polymerase chain reaction, enzymatic assays, flow cytometry) were evaluated in LS180 cells after treatment with different rifampicin or rifabutin concentrations for variable exposure times and eventually normalized to actual intracellular concentrations. In addition, inhibitory effects on CYP3A4 and Pgp activities were investigated. While rifampicin is poorly taken up by LS180 cells, it strongly activates PXR and leads to enhanced expression and activity of CYP3A4 and Pgp. In contrast, rifabutin is a significantly less potent and less efficient PXR activator and gene inducer, despite sixfold to eightfold higher intracellular accumulation. Finally, rifabutin is a potent inhibitor of Pgp (IC50 = 0.3 µM) compared to rifampicin (IC50 = 12.9 µM). Together, rifampicin and rifabutin significantly differ by their effects on the regulation and function of CYP3A4 and Pgp, even when controlled for intracellular concentrations. Rifabutin’s concurrent Pgp inhibitory action might partly compensate the inducing effects, explaining its weaker clinical perpetrator characteristics.

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“…On the other hand, cell proliferation showed sufficient cell proliferation effects at concentrations of 1% to 2% FBS for both CJ and MDCK cells. Interestingly, cell number comparison by CV staining [36] showed that CJ cells stained significantly stronger than MDCK cells (Fig. 4b).…”

Section: Resultsmentioning

confidence: 99%

Characterization of a Unique Spontaneous Calcifying Cell Line (CJ): a Novel Tool for the Study of Ectopic Calcification

Ida,

Yamane

2024

Preprint

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PurposeDue to the lack of an appropriate in vitro evaluation system, there is no effective prevention or treatment for ectopic calcification diseases (ECD). We obtained canine renal adenocarcinoma cells (CJ cells) that spontaneously form large amounts of calcified precipitates (CaP-ppt) and examined whether they could serve as a model for the early stages of ectopic calcification (EC).MethodsDispersed cells obtained by collagenase-DNase digestion were cultured in 10% FBS, antibiotic-containing DMEM-high glucose medium (standard medium). CaP-ppt was stained with Alizarin Red (AR) and colorimetrically quantified after acid extraction. Cell volume was determined by Crystal violet (CV) staining followed by extraction and colorimetric quantification. Calcium (Ca) and phosphate (PO4) were determined with a commercial kit to obtain the Ca/P ratio. Screening of anti-CaP-ppt compounds was performed in the same 96-well plate in the order of cell culture ⇒ CaP-ppt quantification ⇒ cell quantification and evaluated by IC50value.ResultsCJ cells produced large amounts of CaP-ppt on standard medium alone without external phosphate addition; CaP-ppt formation was not accompanied by cell death, but on the contrary, CaP-ppt increased at physiological pH values around pH 7.4 due to active cell metabolism. The Ca2+and PO43-partitioning kinetics to CaP-ppt were found, and the Ca/P ratio of CaP-ppt was stable at 1.35. The anti-Cap-ppt effects of bisphosphonates and all-trans retinoic acid (ATRA) were also confirmed in this CJ cell CaP-ppt system.DiscussionThere was doubt about the vascular calcifying cell model with the addition of high concentrations of phosphoric acid, but there were no suitable alternative cells. Spontaneously calcifying CJ cells provide a fundamental solution to this problem. Anti-CaP-ppt screening also eliminates the need for medium exchange, thus saving labor and cost. The Ca/P ratio of CaP-ppt in CJ cells is 1.35, the same as that of amorphous Ca phosphate (ACP), corresponding to the early (reversible) stage of EC. Therefore, it has favorable conditions as an evaluation system for drug discovery.ConclusionCJ cells, which calcify at physiological phosphate concentrations (0.9 mM) in standard media, are useful and novel research material for basic and preventive studies of ECD and for drug development studies.

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Crystal Violet Staining is a Reliable Alternative to Bicinchoninic Acid Assay-Based Normalization

Cited by 14 publications

References 12 publications

NAV-001, a high-efficacy antibody-drug conjugate targeting mesothelin with improved delivery of a potent payload by counteracting MUC16/CA125 inhibitory effects

NAV-001, a high-efficacy antibody-drug conjugate targeting mesothelin with improved delivery of a potent payload by counteracting MUC16/CA125 inhibitory effects

Comprehensive in vitro analysis evaluating the variable drug–drug interaction risk of rifampicin compared to rifabutin

Characterization of a Unique Spontaneous Calcifying Cell Line (CJ): a Novel Tool for the Study of Ectopic Calcification

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