After cells are transfected with FBXL8-specific siRNA or control scrambled siRNA for 16 h, both Alamar blue and trypan blue dye exclusion were used to measure the cell proliferation. The cell growth was measured by Alamar blue. The metabolic activity of the cells was determined at 570 nm using a Synergy H4 microplate reader (BioTek) at time points indicated. For trypan blue dye exclusion, to assess total cell number, cells were scraped and resuspended in equal volumes of culture medium and trypan blue dye (0.4% solution; Gibco, Waltham, MA, USA) and counted using an improved Neubauer hemocytometer.
Neubauer hemocytometer
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Sweden
The Neubauer hemocytometer is a scientific instrument used for counting and measuring the concentration of cells, such as blood cells or other types of cells, in a liquid sample. It consists of a thick glass slide with a ruled grid pattern that creates a precise counting chamber. The grid pattern allows the user to count the number of cells within a defined volume of the sample, which can then be used to calculate the cell concentration.
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Lab products found in correlation
Trypan blue, by Thermo Fisher Scientific (2 mentions)
Trypan blue dye, by Thermo Fisher Scientific (1 mentions)
Synergy h4 microplate reader, by Agilent Technologies (1 mentions)
Stem cell factor (scf), by STEMCELL (1 mentions)
Trypan blue dye exclusion test, by Thermo Fisher Scientific (1 mentions)
Inverted phase contrast microscope, by Olympus (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
Trypan blue staining, by Thermo Fisher Scientific (1 mentions)
Ficoll paque density gradient centrifugation, by GE Healthcare (1 mentions)
6 protocols using neubauer hemocytometer
1
Quantifying Cell Proliferation and Viability
2
Cell Proliferation Assay Protocol
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For the proliferation assay, cells were seeded in 24‐well plates and cultured for 72 h. The cells were maintained in the exponential phase of growth. The number of cells was counted using an improved Neubauer hemocytometer with trypan blue (Gibco) every 24 h. Three independent experiments were conducted.
3
Expansion of CD34+ Hematopoietic Cells
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Purified CD34+ cells were cultured in DMEM plus 10% FBS supplemented with recombinant cytokines, including SCF, Flt-3 ligand (FL), and TPO (100 ng/mL, all from stem cell technologies, Vancouver, BC, Canada).31 (link) The cells were incubated under a fully humidified incubator at 37℃ with a 5% CO2 atmosphere for 7 days. On day 7, the count and viability of cells were detected by trypan blue dye exclusion test (Gibco, UK) using a Neubauer hemocytometer. Furthermore, the cells were evaluated by flow cytometry for verification of expansion without differentiation.
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Purified CD34+ cells were cultured in DMEM plus 10% FBS supplemented with recombinant cytokines, including SCF, Flt-3 ligand (FL), and TPO (100 ng/mL, all from stem cell technologies, Vancouver, BC, Canada).31 (link) The cells were incubated under a fully humidified incubator at 37℃ with a 5% CO2 atmosphere for 7 days. On day 7, the count and viability of cells were detected by trypan blue dye exclusion test (Gibco, UK) using a Neubauer hemocytometer. Furthermore, the cells were evaluated by flow cytometry for verification of expansion without differentiation.
4
Morphological Features and Viability of SHED
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The morphological features of SHED were observed by an inverted phase-contrast microscope (Olympus, Japan) at the start of each passage. Cells were gently rinsed in Dulbecco's phosphate-buffered saline (DPBS, Gibco) to remove free-floating cells prior to photomicrography. Viability was assessed using 0.4% trypan blue (Gibco) in triplicates by counting in a Neubauer hemocytometer.
5
Mobilizing Peripheral Blood Cells
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Cells were collected from donors who received 10 μg/kg/day of recombinant G-CSF (Filgrastim, Sandoz Biopharmaceuticals, Paris, France) every 12 hours starting five days before collection. Leukapheresis were performed with a COBE Spectra continuous flow blood cell separator (COBE Spectra apheresis system, Caridian BCT, Lakewood, CO, USA). Cell products, anticoagulated with ACD-A, were collected with a 1.1 ml/min flux in a 500 ml container, from which an aliquot of 0.5 ml was used to perform the experiments. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque density gradient centrifugation (GE Healthcare Bio-Sciences, Uppsala, Sweden) and counted in Neubauer hemocytometer using 0.4% trypan blue staining (Gibco, Carlsbad, CA).
6
Proliferation Kinetics of BMSCs
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Proliferation of BMSCs expanded according to different protocols was measured by counting cells with a Trypan Blue exclusion test after 24 h, 48 h, 72 h and 7 days after seeding at p.2. Briefly, cells were seeded at a density of 3000/cm2 in 6-well tissue culture plates and cultured with αMEM or XSFM medium, with or without a fibronectin coating. After cell detachment with 0.05% Trypsin-Ethylenediaminetetraacetic acid (EDTA) (Gibco), the cells were counted in a Neubauer hemocytometer after a two-fold dilution in Trypan Blue.
The population doubling time (PDT) was calculated using the following formula: where t is time (hours), N is the number of cells, and N2 and N1 represent the number of cells at t2 (72 h) and t1 (24 h), respectively. The PDT was calculated in the range 24–72 h for the cells to be in the exponential phase of cell growth.
At 72 h, cell cultures were imaged for a qualitative assessment of cell morphology.
The population doubling time (PDT) was calculated using the following formula: where t is time (hours), N is the number of cells, and N2 and N1 represent the number of cells at t2 (72 h) and t1 (24 h), respectively. The PDT was calculated in the range 24–72 h for the cells to be in the exponential phase of cell growth.
At 72 h, cell cultures were imaged for a qualitative assessment of cell morphology.
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