The Effect of Dithiothreitol on the Transcriptome of Induced Sputum Cells

Goldmann, Torsten; Pedersen, Frauke; Seehase, Sophie; Marwitz, Sebastian; Lang, Dagmar S.; Kirsten, Anne-Marie; Zabel, P.; Vollmer, Ekkehard; Magnussen, H; Rabe, Klaus F.; Watz, Henrik

doi:10.1159/000348392

Cited by 4 publications

(4 citation statements)

References 5 publications

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“…Furthermore, the addition of DTT does not dissolve all mucus clusters. DTT has been found to affect leukocyte surface markers essential for leukocyte subtyping 16 , as well as the presence and concentration of proteins 17 . On the contrary, the mechanical mucus dissolution in the present study completely eliminated mucus clusters compared to the non-dissolved samples or the DTT dissolved samples, showing that the mechanical mucus dissolution allows us to have a sample easier and more appropriate to use for testing.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of airway inflammation in mechanically ventilated patients using cell count and protein concentration

Stamatiou

Tsolaki

Hatziefthimiou

et al. 2021

Sci Rep

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Mechanically ventilated (MV) patients may present airway inflammation and elevated secretion production. However, it is unknown whether cell and/or protein counts in bronchial samples may be useful to evaluate their clinical condition. Our aim was to standardize sampling and propose a new mechanical mucus dissolution in Tracheal-Bronchial secretions. In all patients, bronchial lining fluid aspiration (BLF), Bronchoalveolar lavage (BAL) and Bronchial Washings (BW40, BW5) were performed, while visible bronchial secretions were obtained via bronchoscopy (VBS) and blinded, via a common catheter for tracheobronchial aspiration (AC). Mucus was mechanically or DTT dissolved and cell number was count. Protein, albumin and TNF-α levels were measured, in mucus dissolved samples from control and MV patients. Cell number and protein levels were elevated in mucus dissolved compared to non-dissolved, or DTT dissolved. Cell number and TNF-α levels were elevated in MV patients compared to controls, while protein levels were lower in MV patients. Differences in cell and protein levels were observed in samples acquired using different sampling technics. Therefore, mechanical mucus dissolution provides a proper sample for evaluation, and the sampling technic used can influence the sample’s characteristics.

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Section: Discussionmentioning

confidence: 99%

Evaluation of airway inflammation in mechanically ventilated patients using cell count and protein concentration

Stamatiou

Tsolaki

Hatziefthimiou

et al. 2021

Sci Rep

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“…It has been shown that DTT pretreatment can influence the detection of cytokine protein and gene expression levels in sputum sample. 8,9 Therefore, in our study, all sputum samples were uniformly processed with DTT to allow standardization in this study, but this does not fully overcome the measurement errors due to the DTT introduction itself.…”

Section: Discussionmentioning

confidence: 99%

“…Further technical problems include dithiothreitol (DTT) pretreatment which is used to dissolve disulfide bonds to make sputum less viscous, but can also dissolve the same bonds in several cytokines leading to modified levels. 8,9 Finally, it is conceivable that treatments tested for their effect on sputum inflammation can have an effect by themselves on sputum cytokine concentrations by affecting the water content, such as anticholinergic medication.…”

Section: Introductionmentioning

confidence: 99%

Responsivity and Reproducibility of Sputum Inflammatory Biomarkers During COPD Exacerbation and Stable Phases – A Pilot Study

Ditz¹,

Kistemaker²,

Berge³

et al. 2021

COPD

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“… For example, combine 2 mL of CFASN with 4 mL plain RPMI for a total of 6 mL. An important contrast to other protocols is the exclusion of any denaturing chemicals when processing the sputum, such as dithiothreitol (DTT) which influences cell phenotype and transcriptome ( Goldmann et al., 2013 ; Loppow et al., 2000 ; Makam et al., 2009 ; Tirouvanziam et al., 2008 ). …”

Section: Before You Beginmentioning

confidence: 99%

Mass production of human airway-like neutrophils via transmigration in an organotypic model of human airways

et al. 2021

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Summary Neutrophils are difficult to study, particularly in tissues, due to their short half-life and propensity for activation. We describe an organotypic airway model that uses patient airway fluid to enable the transmigration of blood neutrophils to acquire an airway-like phenotype in order to better understand their contribution to airway diseases. In particular, we showcase how conditioned neutrophils modulate their bacteria-killing abilities. For complete details on the use and execution of this protocol, please refer to Margaroli et al. (2021) .

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The Effect of Dithiothreitol on the Transcriptome of Induced Sputum Cells

Cited by 4 publications

References 5 publications

Evaluation of airway inflammation in mechanically ventilated patients using cell count and protein concentration

Evaluation of airway inflammation in mechanically ventilated patients using cell count and protein concentration

Responsivity and Reproducibility of Sputum Inflammatory Biomarkers During COPD Exacerbation and Stable Phases – A Pilot Study

Mass production of human airway-like neutrophils via transmigration in an organotypic model of human airways

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