For intracellular staining of phosphor-proteins, cells were fixed with 2% paraformaldehyde, followed by permeabilization in 95% methanol. The following antibodies were used: anti-p-PDHE1α (AP1062; Calbiochem, Darmstadt, Germany) and a fluorescein isothiocyanate conjugated antirabbit secondary antibody (Santa Cruz Biotechnology, Dallas, TX). For staining of intracellular cytokines/transcription factors, either the Mouse Foxp3 Buffer Set 560409 (BD Biosciences, Franklin Lakes, NJ) or the Foxp3/Transcription Factor Staining Buffer Set 00-5523-00 (eBioscience, San Diego, CA) was used along with the following antibodies: CD45-BV421, CD3-PECy7, CD4-APC, CD8-Alexafluor700, NK1.1-BV711, IFN-γ-BV510, IL-4-BV605, IL-17-PerCP-Cy5.5, CD25-BV785, Foxp3-FITC, CD69-PerCPCy5.5, and CD62L-PerCPCy5.5 (all from BioLegend, San Diego, CA). Cell viability was measured using a Live/Dead staining kit (Thermo Fisher). Cell death was measured using an FITC/Annexin V Apoptosis Detection Kit (556547; BD Biosciences).
Live dead staining kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany
The Live/Dead staining kit is a laboratory tool used to assess the viability of cell samples. It utilizes fluorescent dyes to differentially stain live and dead cells, enabling visualization and quantification of the cell population.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Lsrii flow cytometer, by Tree Star (4 mentions)
Cck 8, by Dojindo Laboratories (4 mentions)
Lsm 510, by Zeiss (4 mentions)
Ionomycin, by Merck Group (4 mentions)
Hoechst, by Thermo Fisher Scientific (3 mentions)
Calcein am, by Thermo Fisher Scientific (3 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (3 mentions)
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
Alamar blue assay kit, by Thermo Fisher Scientific (3 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Confocal microscope, by Zeiss (2 mentions)
Fluorescence microscope, by Zeiss (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Anti cd16 cd32 fc block, by BD (2 mentions)
Alamar blue kit, by Thermo Fisher Scientific (2 mentions)
2 n morpholino ethanesulfonic acid, by J&K Scientific (2 mentions)
Citric acid, by Merck Group (2 mentions)
Microplate reader, by Bio-Rad (2 mentions)
120 protocols using live dead staining kit
1
Intracellular Staining and Cytokine Detection
2
Microspheres Preparation for Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells and culture media were obtained from PromoCell (Heidelberg, Germany). Culture materials and reagents were obtained from Dominique Dutscher (Brumath, France). ELISA kits were purchased from R&D Systems (Lille, France), Live/Dead® staining kit from Thermo Fisher Scientific (Waltham, MA, USA). For the microspheres preparation, PEG-bis(N-succinimidyl succinate) (PEG-NHS), anhydrous dimethylsulfoxide (DMSO) and SPAN80® were obtained from Sigma-Aldrich (St Louis, MO, USA), and poly(L-lysine) dendrigrafts (DGL) were obtained from COLCOM (Clapiers, France).
3
Encapsulated cell viability and phenotype
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell viability and phenotype were assessed using either a MACSQuant (Miltenyi Biotec, UK) or AttuneNxT (Thermo Scientific, UK) flow cytometer. Cells were harvested from (i) monolayer culture (naked—nTie2-iBMMs) and alginate capsules (encapsulated—eTie2-iBMMs) at days 1, 3, 5, 7, 14 and 21 post-encapsulation and (ii) digested adductor muscle specimens. Cells were washed and FcR receptors blocked using FcR blocking reagent (Miltenyi Biotec, UK). Cell viability was determined using a Live-Dead Staining Kit (Thermo Fisher Scientific, UK) for annexin V and PI according to manufacturer’s instructions. Antibodies for assessment of cell phenotype or muscle cell content are listed in Supplementary Data , Table S4 . All experiments utilised fluorescence minus one controls to determine positive cell surface expression, and analysis of acquired data was carried out using FlowJo V10 software. Gating panels are detailed in Supplementary Data , Figures S5 and S6 .
4
Cell Viability Assay in Irradiated Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In order to analyze cell viability in non-irradiated and irradiated cultures, cells were separated per cell co-culture and cell monocultures. Cells were seeded in five different cultures and irradiated after 24 h with one dose of 5 Gy. Living/dead cells were detected with Calcein AM (1 μg/mL) and propidium iodide (2 μg/mL). The Live/dead staining Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used according to the manufacturer’s instructions. Photographs were taken with an Axio Observer Z.1 fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany). Proportional bar graphs were performed with Microsoft Excel version 16.0 (Microsoft Corporation, Redmond, WA, USA).
5
Multiparametric Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Results were reported as absolute numbers of T cells per organs after performing an initial organ cell count using trypan blue and staining and acquiring a fixed number of cells. One million splenocytes or lung cells were stained using antibodies to CD3 (564380), CD8 (551162), CD4 (561025), CD62L (553150), CD44 (562464), CCR7 (12-1971-80), CD103 (562772), CD69 (740220), CD11a (740849), Live/Dead Fixable Aqua Dead Cell Stain Kit (L34957). All the antibodies were purchased by BD Biosciences. CCR7 antibody was purchased from eBiosciences and Live/Dead staining kit was purchased from Thermo Fisher Scientific. After fixation and storage at 4°C overnight, the cells were analyzed using a BD Fortessa flow cytometer. The FlowJo software program was used to analyze the data (Tree Star Inc., Stanford, CA).
For intracellular staining, cells were fixed and permeabilized according to manufacture instruction of using Fixation/Permeabilization Solution Kit with BD GolgiStop (554715: BD Biosciences) and stained for IFN-γ (564336: BD Biosciences) and IL-2 (560547: BD Biosciences). All staining antibodies were used at 1:200 or 1:300 or 1:500 dilution depending on the experiment. Sample acquisition was performed on a BD Fortessa flow cytometer using FACS Diva software followed by data analysis with FlowJo software.
For intracellular staining, cells were fixed and permeabilized according to manufacture instruction of using Fixation/Permeabilization Solution Kit with BD GolgiStop (554715: BD Biosciences) and stained for IFN-γ (564336: BD Biosciences) and IL-2 (560547: BD Biosciences). All staining antibodies were used at 1:200 or 1:300 or 1:500 dilution depending on the experiment. Sample acquisition was performed on a BD Fortessa flow cytometer using FACS Diva software followed by data analysis with FlowJo software.
6
Assessing H. pylori Viability via Live/Dead Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine the viability of H. pylori strains when assessing the survival at specific time points, the study was expanded to include fluorescence analysis using Live/Dead staining kit (L7012, ThermoFisher, Waltham, MA, USA) [38 (link)]. Slides were viewed under an Olympus BX51 microscope (Olympus Optical, Tokyo, Japan), working with a ×10 lens. Using the ImageJ, the green and red fluorescence of regions of interests (ROIs) (50 ROIs/tested sample) was determined and presented as the mean green/red fluorescence ratio. The fluorescence intensity of SYTO9 (green fluorescent dye) and propidium iodide (red fluorescent dye) was measured at emission levels of 530 nm and 640 nm, respectively.
7
Assessment of Cell Viability and Death
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cell viability was assessed using the LIVE/DEAD staining kit (Thermo Fisher Scientific). The neuronal channel of the Brain-Chips was incubated for 30 min in PBS containing 1 μM calcein AM and 2 μM ethidium homodimer-1 (EthD1). The channel was then washed with PBS and imaged under a motorized fluorescent microscope (Zeiss confocal microscope). Four frames per chip at ×10 magnification were selected for each treatment, and particles were counted using Fiji/ImageJ. Data were expressed as the average live cells/total number of cells (sum of calcein-AM positive and ethidium homodimer positive). In order to confirm the efficiency and reliability of this assay, we set up a positive control (DMSO treatment) and negative control (no treatment) to do the parallel experiment with the αSyn treatment. In cell death analysis, TUNEL-positive cell density was measured for each treatment, and particles were counted using Fiji/ImageJ.
8
Comprehensive Cell Viability Assessment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell viability was assessed by the Live/Dead staining kit (Thermo Fisher Scientific) and CellTiter-Glo 3D cell viability assay (Promega). The CPCs cultured in Matrigel were released using Cell Recovery Solution (Corning), and cells cultured in the hybrid hydrogel were released using GelMA lysis solution. The cells in the organoids were then incubated with 1 μl of Live/Dead reagent (an indicator of dead cells) in the expansion medium for 30 min and then imaged under a Leica DMi8 microscope with 488/570-nm excitation wavelength. The number of dead cells was counted per organoid using ImageJ (National Institutes of Health, USA). For the CellTiter-Glo 3D cell viability assay, the collected cells were incubated with the working fluid for 10 min, and then the cell suspensions were transferred into a black light-proof 96-well plate. Cell viability was recorded by a Tecan M1000 reader according to the manufacturer’s protocol.
9
Cytotoxicity Evaluation of Extract
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HGFs were seeded at a density of 105 cells/well in 24-well plate. The extract from each group was added after 4 h of proliferation. After 24 h of incubation, a live/dead staining kit (L10119, Thermo Fisher Scientific Inc, USA) was utilized to assess the cytotoxicity of the extracts. Calcein-AM was a staining reagent for fluorescently labeling living cells with green fluorescence, and its working concentration was 1 μM. In addition, PI (3 μM) only stained dead cells and excited red fluorescence. Finally, dyed cells were visualized through inverted epifluorescence microscope.
10
Quantifying Cell Viability in Tissue Constructs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In order to determine the cell viability after 28 days of culture, the constructs were first washed in Dulbecco’s phosphate buffered saline (DPBS, Gibco) before being stained with a Live/Dead staining kit (ThermoScientific) according to the manufacturer’s protocol. Images were acquired on a Cytation 5 multi-mode reader using Gen5 imaging software (BioTek, Winooski, VT, USA). The area of positive calcein stain was determined in two fields per sample using a custom MATLAB code utilizing MATLAB R2020a software (Figures S1 and S2 ).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!