Cell growth-rate was measured by crystal violet (C6158, Sigma Aldrich, Overijse, Belgium). This method allows the assessment of cellular cytotoxicity following a genetic or pharmacological treatment. After treatment, living cells remain adherent to the well and can be stained with crystal violet who binds ribose molecules on DNA. The number of cells stained positively with crystal violet is proportional to the number of living cells. First, cells were fixed with a solution of 10% methanol (20847.307, VWR, Leuven, Belgium) and 10% acetic acid (84874.290, VWR, Leuven, Belgium) for 15 min at room temperature (RT). Then, the plate was dried and stained with 0.5% crystal violet solution for 15 min at RT. Finally, the plate was washed three times with tap water and let dry. The next day, absorbance was measured at 595 nm by adding 10% acetic acid solution into each well. In select experiments, cells were dissociated with trypsin (25050-014, Life Technologies, Merelbeke, Belgium), and the number of cells was estimated using the automated counter TC20TM (Bio-Rad, Temse, Belgium).
Tc20
Manufactured by Bio-Rad
Sourced in United States, Germany
The TC20TM Automated Cell Counter is a compact and automated cell counting device designed for accurate and reproducible cell counting. It utilizes advanced optics and image analysis technology to provide rapid cell counts and cell viability measurements. The TC20TM is a versatile instrument suitable for a wide range of cell types and applications.
Automatically generated - may contain errors
Lab products found in correlation
Multiskan fc, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Crystal violet, by Merck Group (1 mentions)
C6158, by Merck Group (1 mentions)
Trypsin edta solution, by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Trypan blue dye, by Bio-Rad (1 mentions)
T25 flask, by Greiner (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
M3434, by STEMCELL (1 mentions)
Pacap38, by Peptide Institute (1 mentions)
Eclipse te300, by Nikon (1 mentions)
Collagenase type 2, by Thermo Fisher Scientific (1 mentions)
T8154, by Merck Group (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Cck 8 reagent, by Solarbio (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Bio-Rad (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
27 protocols using tc20
1
Crystal Violet Cytotoxicity Assay
2
Equine Mesenchymal Stem Cell Expansion
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ASCs-EXP and ASCs-SVF of the abd, rb and sc localizations from n = 3 horses were included in the experiment. On day zero, 30,000 cells/well of P3 from each source and isolation procedure were seeded in 12-well cell culture plates. According to the protocol of Alipour et al. [60 (link)], cells were detached daily after 24 h of cultivation from three wells with Trypsin-EDTA (Trypsin-EDTA solution, 0.25% Trypsin/0.02% EDTA, Sigma Aldrich, Taufkirchen, Germany) and were counted in duplicate using the automated cell counter TC20TM (Bio-Rad Laboratories GmbH, Feldkirchen, Germany). Population doubling time (PDT) was determined using the formula PDT = (T−T0) lg2/(lgNt–lgN0) according to Alipour et al., where T is the harvest time of culture, T0 the starting time of culture, and Nt and N0 are the cell count of harvest and cell count of seeding, respectively [60 (link)].
3
Cell Viability Assessment via Trypan Blue
4
Cell-Free Viral Supernatant Infection
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Cell-free supernatant used in infection was derived as for r, except that 1 day before harvesting of the viral supernatant from infected cells, infected cells were washed twice with PBS and serum-free growth medium added. At the same time, the target cells for the infection were washed twice with PBS and serum-free growth medium added. Cells were split into two wells, and EFV to a final concentration of 40 nM was added to one of the wells. Supernatant was harvested and filtered as described for r, and added to cells at a 1:2 dilution. A fraction of the cells were sorted into lysis buffer at one cell per well 1 day post-infection, split over four wells, and PCR performed as described above to determine HIV copy number per cell. The remaining cells from the same infection were used to determine the frequency of live and dead cells two days post-infection. The concentration of live cells was measured using the TC20TM automated cell counter (Bio-Rad) with trypan blue staining (Lonza).
5
Isolation and Characterization of Human PBMCs
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Peripheral blood (20 mL) was collected in sodium heparin tubes and processed within 8 hours. PBMC were isolated by density gradient centrifugation at 800×g for 20 minutes. The PBMC were washed 5 times with RPMI at 500×g for five minutes. Viability was assessed by trypan blue exclusion; live cells were counted using a Bio-Rad TC20TM automated cell counter. Cells were incubated in RPMI with 10% fetal bovine serum (R10) at 37°C with 5% CO2 until use in the ELISpot assay.
6
Differentiation of PC-12 Cells with NGF
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PC-12 cells were cultured in T25 flasks (Greiner Bio-One GmbH, cat. No. 690 170) in a humidified atmosphere containing 5% CO2 at 37 °C in high-glucose DMEM, supplemented with 10% heat-inactivated HS (Sigma-Aldrich, cat no. H1138), 5% FBS (Sigma-Aldrich, cat. No. F2442), 100 U/mL penicillin, and 50 µg/mL gentamicin sulfate. To induce differentiation, PC-12 cells were incubated in low serum-containing DMEM (1% HS and 1% FBS), supplemented with 100 ng/mL NGF for 5 days. The NGF-containing medium was replaced every other day. The cells were scored as differentiated if the neurites were longer than twice the diameter of one cell body. Cell viability was measured by staining cells with trypan blue dye (Bio-Rad, cat no. 145-0013) and using an automated cell counter (TC 20TM; Bio-Rad, USA) to count live cells. Average neurite length was measured using Image J software (https://imagej.net/ImageJ ).
7
Hematopoietic Stem Cell Assay with PACAP38
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BM mononuclear cells (280,000 cells), CD34+ cells (32,000 cells) or CD34+/SCA1+ cells (8,000 cells) were mixed with 4 mL semi-solid methylcellulose cultures supplemented with the recombinant cytokines mouse interleukin-3, mouse stem cell-derived factor 1, human interleukin-6, and human erythropoietin (M3434, StemCell Technologies, Vancouver, BC)24 (link). The cell suspensions including PACAP38 (2 × 10−14 to 10−6 M, Peptide Institute, Osaka, Japan) were divided into 3 wells of a 6-well plate and cultured for 10 days at 37 °C and 95% humidity in an incubator containing 5% CO2 in air. In the antagonist experiments, a five-fold higher concentration of PACAP 6–38 (Peptide Institute) or VIP 6–28 (Abcam, Cambridge, CA) was added to the medium 60 min before addition of PACAP or VIP. The number of CFU-GM colonies was counted in a 2 × 2 cm area of STEMgrid™-6 (StemCell Technologies) under light microscopy (Nikon Eclipse TE300, Tokyo, Japan) and photographed with a Stylus TG-3 camera (Olympus, Tokyo, Japan) in microscopy mod. Cells in the methylcellulose-based media were repeatedly washed and centrifuged, and finally resuspended in 1.0 mL PBS for cell counting using an automated cell counter (TC20TM, Bio-Rad, Hercules, CA).
8
Cell Viability Assay Protocol
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Two days before treatment, the cells were seeded in 1 × 104 cells/mL in 96-well culture plates. The cell numbers were measured using TC-20TM (Bio-Rad Laboratories, Inc., Hercules, CA, USA). After 1 h of treatment, the cells were cultured for three days. Cell viability was evaluated using a cell counting kit-8 (Dojin, Kumamoto, Japan), according to the manufacturer’s protocol. The absorbance was measured at 450 nm using a MultiSkan FC (Thermo Fisher Scientific).
9
5-aza-dC Dose-dependent Epigenetic Modulation
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OS cell lines were seeded (MG63, 1 × 104; HOS, 2 × 103; 143B, 2 × 103; and MNNG/HOS, 1 × 103 cells) in a 6-well plate on day 0, and treated with various concentrations (0.1, 0.3, 1, 3, 10 μM) of 5-aza-dC on days 1 and 3. To facilitate cell proliferation, which can result in efficient DNA demethylation26 (link), the medium was replaced by a fresh one without 5-aza-dC on day 5, and cells were harvested on day 9. Cell numbers were counted using an automated cell counter (TC20TM, Bio-Rad Laboratories, Inc., CA, USA).
10
Isolation and Characterization of Rabbit Cells
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The cells were retrieved through the following procedure. Tissues from the rabbit skulls were placed in a 1.5 mL tube and centrifuged. After removing the supernatant, the cells were washed with phosphate-buffered saline and then treated with collagenase type II (Gibco BRL) at 1 mg/mL for groups 2, 3, and 4, which contained collagen membranes. The cells were incubated for 30 minutes to 1 hour to dissolve the collagen. All tissues were centrifuged twice at 1,200 rpm for 5 minutes. The cells were then mixed with 9 mL of distilled water and 1 mL of ×10 PBS (Gibco BRL) for 15 seconds and then centrifuged at 1,700 rpm for 6 minutes to hemolyze the red blood cells. After removing the supernatant, 1 mL of PBS and 1 mL of trypan blue solution (T-8154, Merck KGaA, Darmstadt, Germany) were mixed. Next, the number of total cells, the number of live cells, and the ratio of live cells were analyzed using an automated cell counter (TC 20TM, Bio-Rad Laboratories, Berkeley, CA, USA). Before transplantation, the fluorescent-stained stem cells were observed with an inverted microscope (IX71; Olympus Co., Tokyo, Japan) and were photographed with a digital camera (DP70; Olympus Co.). A quantitative evaluation was performed by comparing and analyzing the measured values for each defect.
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