A Convenient Technique to Fix Suspension Cells on a Coverslip for Microscopy

Mihara, Kazuhiro; Nakayama, Tomofumi; Saitoh, Hisato

doi:10.1002/0471143030.cb0430s68

Cited by 10 publications

(9 citation statements)

References 10 publications

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“…As a result, PFA (4%) fixation after 0.2% trypan blue staining was included in this modified protocol. Paraformaldehyde is the polymerized form of formaldehyde and the most effective fixative commonly used in staining protocols [9][10][11][12][13]. Because PFA does not only produce cross-links between proteins but can also fasten proteins into impenetrable network of cellular components.…”

Section: Resultsmentioning

confidence: 99%

Development of a Direct Trypan Blue Exclusion Method to Detect Cell Viability of Adherent Cells into ELISA Plates

Uzuner¹

2018

Celal Bayar Üniversitesi Fen Bilimleri Dergisi

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Cell viability detection is important in cell culture applications including measurement of cell proliferation i.e for understanding cytotoxic effects of compounds on cells. There are some cell viability methods based on fluorescence or non-fluorescence detection. More simplified evaluation for cell viability, such as trypan blue staining, can be preferred before performing fluorescence assays. This appears advantageous when to have a large number of cell samples in ELISA plates after treatments with different concentrations of drug candidates. Thus, further fluorescence assays can include less concentrations rather than experiencing all used along 96well plates. For this, trypan blue exclusion method is an option. Traditionally, treated cells are harvested by centrifugation and incubated with trypan blue within tubes followed by transferring the mixture into a hemacytometer with two chambers and assessed under the microscope. Nevertheless, using a hemacytometer limits practicability of this method when analyzing various cell samples into 96-well plates at the same time. This study was aimed to adapt trypan blue method to in situ staining of adherent cells cultured on ELISA plates. For this, cells were fixed with different fixatives after trypan blue incubation to maintain cells in impenetrable meshwork, and paraformaldehyde was the most effective fixative. This modified protocol was validated by testing the effect of dimethylsulfoxide-a cytotoxic agent-on cells, and expectedly found that cell viability reduced with higher concentrations of dimethylsulfoxide suggesting that in situ detection of cell viability by trypan blue can be a useful tool for preliminary detection of cells cultured on ELISA plates before performing automatized experiments with such flow cytometer and/or microplate reader.

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Section: Resultsmentioning

confidence: 99%

Development of a Direct Trypan Blue Exclusion Method to Detect Cell Viability of Adherent Cells into ELISA Plates

Uzuner¹

2018

Celal Bayar Üniversitesi Fen Bilimleri Dergisi

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“…The cell immobilization on the coverslip was performed as described previously (Nakayama et al, ; Mihara et al, ). Fixation of cells was performed using 4% paraformaldehyde–phosphate‐buffered saline (PBS) solution for 15 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Nuclear extrusion precedes discharge of genomic DNA fibers during tunicamycin‐induced neutrophil extracellular trap‐osis (NETosis)‐like cell death in cultured human leukemia cells

Nakayama

Saitoh

Morotomi-Yano

et al. 2016

Cell Biology International

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We previously reported that the nucleoside antibiotic tunicamycin (TN), a protein glycosylation inhibitor triggering unfolded protein response (UPR), induced neutrophil extracellular trap-osis (NETosis)-like cellular suicide and, thus, discharged genomic DNA fibers to extracellular spaces in a range of human myeloid cell lines under serum-free conditions. In this study, we further evaluated the effect of TN on human promyelocytic leukemia HL-60 cells using time-lapse microscopy. Our assay revealed a previously unappreciated early event induced by TN-exposure, in which, at 30-60 min after TN addition, the cells extruded their nuclei into the extracellular space, followed by discharge of DNA fibers to form NET-like structures. Intriguingly, neither nuclear extrusion nor DNA discharge was observed when cells were exposed to inducers of UPR, such as brefeldin A, thapsigargin, or dithiothreitol. Our findings revealed novel nuclear dynamics during TN-induced NETosis-like cellular suicide in HL-60 cells and suggested that the toxicological effect of TN on nuclear extrusion and DNA discharge was not a simple UPR.

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“…Cells were stimulated with 1 µM alltrans retinoic acid (ATRA) (Sigma) for 5 days to differentiate into neutrophil-like cells (dHL60) (32,33). To confirm dHL60 cell differentiation, ATRA-induced HL60 cells were resuspended in FBS-free RPMI 1640 medium and allowed to settle for 20 min for differentiated dHL60 cells to adhere on round glass slides in a 24-well plate, followed by fixing in 4% paraformaldehyde for 40 min (34). Adhered cells were washed with PBS and stained with DAPI dye (Zhongshanjinqiao, Beijing, China) for observation of lobulated nuclei.…”

Section: Culture and Differentiation Of Hl60 Cellsmentioning

confidence: 99%

Trichinella spiralis Calreticulin S-Domain Binds to Human Complement C1q to Interfere With C1q-Mediated Immune Functions

et al. 2020

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Helminths develop strategies to escape host immune responses that facilitate their survival in the hostile host immune environment. Trichinella spiralis, a tissue-dwelling nematode, has developed a sophisticated strategy to escape complement attack. Our previous study demonstrated that T. spiralis secretes calreticulin (TsCRT) to inhibit host classical complement activation through binding to C1q; however, the C1q binding site in TsCRT and the specific mechanism involved with complement-related immune evasion remains unknown. Using molecular docking modeling and fragment expression, we determined that TsCRT-S, a 153-aa domain of TsCRT, is responsible for C1q binding. Recombinant TsCRT-S protein expressed in Escherichia coli had the same capacity to bind and inhibit human C1q-induced complement and neutrophil activation, as full-length TsCRT. TsCRT-S inhibited neutrophil reactive oxygen species and elastase release by binding to C1q and reduced neutrophil killing of newborn T. spiralis larvae. Binding of TsCRT-S to C1q also inhibited formation of neutrophil extracellular traps (NETs), which are involved in autoimmune pathologies and have yet to be therapeutically targeted. These findings provide evidence that the TsCRT-S fragment, rather than the full-length TsCRT, is a potential target for vaccine or therapeutic development for trichinellosis, as well as for complement-related autoimmune disease therapies.

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A Convenient Technique to Fix Suspension Cells on a Coverslip for Microscopy

Cited by 10 publications

References 10 publications

Development of a Direct Trypan Blue Exclusion Method to Detect Cell Viability of Adherent Cells into ELISA Plates

Development of a Direct Trypan Blue Exclusion Method to Detect Cell Viability of Adherent Cells into ELISA Plates

Nuclear extrusion precedes discharge of genomic DNA fibers during tunicamycin‐induced neutrophil extracellular trap‐osis (NETosis)‐like cell death in cultured human leukemia cells

Trichinella spiralis Calreticulin S-Domain Binds to Human Complement C1q to Interfere With C1q-Mediated Immune Functions

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