Impact of cell harvesting methods on detection of cell surface proteins and apoptotic markers

Abstract

Assays based on the flow cytometry technique allow a convenient analysis of multiple cellular parameters; however, their results should be interpreted cautiously due to a strong impact of confounding factors. Different techniques in cell culturing such as either enzymatic or mechanic detachment of adherent cells can heavily influence the structure of the cell membrane or presence of the surface antigens leading to strong false positive signals, and finally, substantial experimental bias. The aim of our study was to assess and compare the impact of cell harvesting methods (both enzymatic and non-enzymatic) on the apoptosis process and on the surface antigen cytometric analyses. We found significant differences in the quality of analysis in terms of the amount of detected surface markers determined by the detachment method. Our results demonstrated clearly how important it is to carefully choose the appropriate detachment method and may help to avoid mistakes in experiment planning. In conclusion, we recommend to adjust the detachment method to the type of analyzed markers (surface antigens or translocated phosphatidylserine).

CSPG4 as a target for the specific killing of triple-negative breast cancer cells by a recombinant SNAP-tag-based antibody-auristatin F drug conjugate

“…In the same line, long-term serial passages of cell lines can induce genotypic and phenotypic variations in the cell population (Kaur and Dufour 2012 ), which can rise to further heterogeneity and inconsistencies in the data set. Similarly, different cell detachment methods have been reported to cause a discrepancy in cell-surface receptor expression between experiments or studies (Lai et al 2022 ; Nowak-Terpiłowska et al 2021 ) For example, trypsinization, a method characterized by the proteolytic activity of trypsin enzyme, is mostly used for cellular dissociation and detachment. However, this cell detachment method has been found to significantly degrade cell-surface proteins and the extracellular matrix (Lai et al 2022 ; Nowak-Terpiłowskaet al 2021 ).…”

“…Similarly, different cell detachment methods have been reported to cause a discrepancy in cell-surface receptor expression between experiments or studies (Lai et al 2022 ; Nowak-Terpiłowska et al 2021 ) For example, trypsinization, a method characterized by the proteolytic activity of trypsin enzyme, is mostly used for cellular dissociation and detachment. However, this cell detachment method has been found to significantly degrade cell-surface proteins and the extracellular matrix (Lai et al 2022 ; Nowak-Terpiłowskaet al 2021 ). Taking this into consideration, accutase was used throughout this study in virtue of its limited proteolytic activity to a small number of cell-surface proteins, such as CD163 and CD206 (Lai et al 2022 ; Nowak-Terpiłowska et al 2021 ).…”

“…However, this cell detachment method has been found to significantly degrade cell-surface proteins and the extracellular matrix (Lai et al 2022 ; Nowak-Terpiłowskaet al 2021 ). Taking this into consideration, accutase was used throughout this study in virtue of its limited proteolytic activity to a small number of cell-surface proteins, such as CD163 and CD206 (Lai et al 2022 ; Nowak-Terpiłowska et al 2021 ). Yet, accutase was recently reported to temporary suppress both FasL and Fas receptors, which could bias the interpretation of the cell death-related experimental results, given the prominent role they play in apoptosis induction (Lai et al 2022 ).…”

Assessment of chemotherapeutic effects on cancer cells using adhesion noise spectroscopy

“…We collected two samples for each chip: 1) cells attached to the chip and 2) cells floating in the culture medium (i.e., supernatant). The first cellular fraction was detached from the chip using 100 µL Accutase (Sigma-Aldrich GmbH) as a mild-acting enzyme ( Quan et al, 2013 ; Chen et al, 2015 ; Skog et al, 2019 ; Nowak-Terpiłowska et al, 2021 ; Lai et al, 2022 ) after 10 min incubation at room temperature. The second cellular fraction was isolated from the medium.…”

Optical sensor reveals the hidden influence of cell dissociation on adhesion measurements

“…Proteolytic enzymes act by digesting extracellular proteins, and components of the ECM, the adhesion complexes, and partially the glycocalyx 17 . The superiority of trypsin treatment over the mechanical methods seems established 18 moreover scraping results in lower viability while increasing the amount of necrotic and apoptotic cells 19 , 20 .The regularly used 0.05% trypsin for 2 min protocol for cell dissociation does not lead to complete removal of the glycocalyx 21 . However, the density of the glycocalyx seems to contribute to the fine morphology of the plasma membrane 22 .…”