In vitro studies with CAR-T cells (CD19-CD28z and CD19-BBz) were performed for the assessment of efficacy and cytotoxic capacity. For that purpose, anti-CD19-expressing CAR-T cells and CD19-expressing RAJI cells were cultured for 24 h, 7 days, and 14 days (CAR-T: RAJI; 1:1, 5:1, 10:1). Anti-cancer profiling studies on days 14 and 21 were analyzed using CD3-PC7 (Beckman Coulter, 6607100), CD4-APC-A700 (Beckman Coulter, B10824), CD8-PC5.5 (Beckman Coulter, B21205), EGFR-A488 (R&D Systems, FAB10951G), CD19-ECD (Beckman Coulter, A07770), CD25-APC (Miltenyi Biotech, 130-113-284), and CD107a (LAMP-1) -PE (Miltenyi Biotech, 130-111-621) by Cytoflex Flow Cytometer analysis. In the co-culture experiments, the death of CD19+ RAJI cells after 48 h and the CD25 activation (IL2RA, IL-2 receptor alpha chain) and CD107a (marker for degranulation of lymphocytes) cytotoxic de-granulation biomarkers of CAR-T cells and control T cells in CD3+ T cells were analyzed by flow cytometry. Survival analysis of CAR-T cells was performed to control the cell viability of RAJI cells and CAR-T cells. Trypan blue (Biological Industries, #03-102-1B) was applied to identify and count surviving cells. Cell counting and viability analysis were performed with the BIO-RAD TC20 Automated Cell Counter.
Trypan blue
Manufactured by Sartorius
Sourced in Israel, Germany
Trypan blue is a vital dye used in cell counting and viability assays. It is a blue azo dye that selectively penetrates the cell membrane of dead or dying cells, coloring them blue. This allows for the differentiation between live and dead cells under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Sartorius (4 mentions)
Penicillin streptomycin, by Sartorius (3 mentions)
Trypsin, by Sartorius (3 mentions)
Sytox green, by Thermo Fisher Scientific (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
B rker chamber, by Sartorius (2 mentions)
Rpmi 1640, by Sartorius (2 mentions)
Protease inhibitors cocktail, by Roche (2 mentions)
Glutamine, by Sartorius (2 mentions)
Streptomycin, by Sartorius (2 mentions)
Fetal bovine serum (fbs), by Sartorius (2 mentions)
Tc20 automated cell counter, by Bio-Rad (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Cd3 pc7, by Beckman Coulter (1 mentions)
Cd4 apc a700, by Beckman Coulter (1 mentions)
Cd8 pc5, by Beckman Coulter (1 mentions)
Egfr a488, by R&D Systems (1 mentions)
27 protocols using trypan blue
1
In Vitro Assessment of CAR-T Cell Efficacy
2
Neutrophil Activation Assay with Bioactive Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cytochalasin B (CB), dextran, dimethyl sulfoxide (DMSO), ethyl acetate, N-formyl-methionyl-leucyl-phenylalanine (fMLF), n-hexane, methanol, paraformaldehyde, and superoxide dismutase were obtained from Sigma-Aldrich (St. Louis, MO, USA). Hank’s balanced salt solution (HBSS), Hoechst 33342, and SYTOX Green were acquired from Thermo Fisher Scientific (Waltham, MA, USA). Trypan blue was purchased from Biological Industries (Beth Haemek, Israel). Ficoll-Paque Plus was procured from GE Healthcare (Little Halfont, Buckinghamshire, UK). Mouse monoclonal antibodies to neutrophil elastase were purchased from Millipore (MABS46, MA, US), and rabbit polyclonal antibodies to histone H3 were purchased from Abcam (ab5103, Cambridge, UK). Isoorientin, orientin, and cepharanthine were purchased from Sunhank Technology Co.,ltd (Tainan, Taiwan).
3
Cellular Uptake and Cytotoxicity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were counted using trypan blue (Biological Industries), and 0.1 × 106 cells were placed in tissue culture 12-well plates (Greiner Bio-One, Germany) with 1 ml of growing medium. MC3 or c-LNPs were added to the wells at RNA amounts of 0.1 to 2 μg. Cells were incubated with the treatments in standard culture conditions for 24 to 120 hours. Then, cells were washed three times, incubated in a fresh culture medium, and collected for flow cytometry (72 to 96 hours) or cell cycle assays (24 to 48 hours), as described below. For 005 cells, cLNPs were preincubated with ApoE3 (0.001 mg/ml; PeproTech, USA) before the addition to the cells.
4
Quantitative Analysis of hASC Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
3 × 103 hASC and DFAT were seeded in 12 well format plates (Sigma-Aldrich) in GM. Every 24 h for one week; cells were trypsinized with Trypsin EDTA (Biowest), centrifuged at 1200 rpm for 10 min at 22 °C, suspended in GM and counted with Burker’s chamber after Trypan Blue (Biological Industries, Cromwell, CT, USA) staining at a dilution 1:1 with PBS. The microscopic observation allowed us to perform a quantitative analysis: the number of viable cells per milliliter was expressed as the difference between the total cell count and the number of non-viable cells per milliliter. The viable cell count was expressed as the mean of two independent counts.
Cell generation time (g) was calculated by the function: g = t/n where: t = 1; n = (Log N−Log N0)/(Log 2); N = cells number; N0 = initial cell number.
Cell generation time (g) was calculated by the function: g = t/n where: t = 1; n = (Log N−Log N0)/(Log 2); N = cells number; N0 = initial cell number.
5
Adalimumab Effects on NHDF Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The NHDF cell line (fourth passage) was cultured in the FBM medium (Fibroblast Basal Medium; Lonza, Basel, Switzerland), supplemented with hFGF-B (Human Fibroblast Growth Factor-Basic) and insulin and gentamicin (FGM™ SingleQuots™; Lonza, Basel, Switzerland) at 37°C in a 5% CO2 incubator (Direct Heat CO2; Thermo Scientific, Waltham, MA, USA). The quantity of cells and their viability were monitored by cell counting in the Bürker chamber after staining them with 0.2% trypan blue (Biological Industries, Beit HaEmek, Israel). To test the effects of the drug on gene expression, 8 μg/ml of adalimumab (AbbVie Biotechnology GmbH, Knollstrasse, Germany) was added to the cell culture for 2, 8, and 24 hours. Control cells were not treated with adalimumab. The concentration was customized to the level of adalimumab in the bodies of psoriatic patients.
6
In Vitro Psoriasis Model: LPS-Induced Inflammation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HaCaT cells (CLS Cell Lines Service, Eppelheim, Germany) were cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 4500 mg/L glucose (Sigma-Aldrich), 10% fetal bovine serum (Sigma-Aldrich), 100 U/mL penicillin (Sigma-Aldrich), 100 mg/mL streptomycin (Sigma-Aldrich), and 2 mM glutamine (Sigma-Aldrich) at 37°C and 5% CO2. Based on our previous studies [15–17 (link)], HaCaT cells were incubated with 1 μg/mL LPS for 8 h, followed by incubation with 8 μg/mL adalimumab or 100 ng/mL of CSA for 2 (H_2 group), 8 (H_8 group), or 24 h (H_24 group). HaCaT cells not treated with LPS, adalimumab, or CSA served as controls. The concentrations of the drugs were customized according to those observed in the serum of patients with psoriasis.
The concentrations of the drugs were customized according to those observed in the serum of patients with psoriasis. The quantity of cells and their viability were monitored by cell counting in the Bürker chamber after staining them with 0.2% trypan blue (Biological Industries, Beit HaEmek, Israel).
The concentrations of the drugs were customized according to those observed in the serum of patients with psoriasis. The quantity of cells and their viability were monitored by cell counting in the Bürker chamber after staining them with 0.2% trypan blue (Biological Industries, Beit HaEmek, Israel).
7
Optimized Immunomodulatory Peptide Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following reagents were purchased as detailed: acrylamide/bisacrylamide, LPS (from E. coli, clone 055:B5), TEMED, and Tween-20 from Sigma-Aldrich Israel Ltd. (Rehovot, Israel); anti-rabbit horseradish peroxidase-conjugated IgG from Jackson Immuno Research Laboratories (West Grove, PA, USA); EZ-ECL, fetal calf serum, L-glutamine, penicillin-streptomycin, RPMI 1640 and trypan blue from Biological Industries (Kibbutz Beit Haemek, Israel); ELISA kits for human IL-10 and TGF-β from BioLegend (San Diego, CA, USA); ELISA kits for human TNF-α and IL-6 from BD Bioscience (San Jose, CA, USA); and protease inhibitors cocktail was purchase from Roche (Basel, Switzerland). FKD, FKE, FKECH, FKECHLA, YKE, and EKF peptides were synthesized by GL Biochem (Shanghai) Ltd. (Shanghai, China) with purity typically over 90%.
8
Cell Counting and Viability Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell number and viability were evaluated by the NucleoCounter NC-200 Automated Cell Counter (ChemoMetec, Denmark). Results were verified by a manual count following a Trypan blue staining (Biological Industries, Israel).
9
Cell Culture and Characterization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Monolayer-adherent HEK-293T cells (transformed human embryonic kidney cells) and SY5Y-SH cells (bone-marrow-derived human neuroblastoma cells) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco by Thermo Fisher Scientific, USA) supplemented with 10% (vol/vol) fetal bovine serum (FBS) (Thermo Fisher Scientific, USA) and 0.01% (vol/vol) pen–strep solution ×10 (Biological Industries, Israel).
Cells were incubated at 37 °C in a 5% CO2 atmosphere. Before use, each cell line was confirmed to have no mycoplasma contamination using the EZ-PCR Mycoplasma Detection Kit (Biological Industries, Israel).
Prior to each experiment, the cells were dissociated using trypsin 0.25% EDTA (Thermo Fisher Scientific, Israel), stained with trypan blue (Biological Industries, Israel), and counted using the CountessTM II Automated Cell Counter (Invitrogen at Thermo Fisher Scientific, USA).
Cells were incubated at 37 °C in a 5% CO2 atmosphere. Before use, each cell line was confirmed to have no mycoplasma contamination using the EZ-PCR Mycoplasma Detection Kit (Biological Industries, Israel).
Prior to each experiment, the cells were dissociated using trypsin 0.25% EDTA (Thermo Fisher Scientific, Israel), stained with trypan blue (Biological Industries, Israel), and counted using the CountessTM II Automated Cell Counter (Invitrogen at Thermo Fisher Scientific, USA).
10
Trypan Blue Cell Viability Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Trypan blue (Biological Industries, Beit Haemek, Israel) was mixed in a 1:1 ratio with the cells. Then the cells were counted using automatic cell counter (NanoEntek, Waltham, MA, USA). Live cells remained unstained, while dead cells assimilated the dye. The cell counter counted the number of dyed and undyed cells in each sample. Using these values, it calculated the percentages of dead cells in the culture.
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!