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Neutral red

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Neutral red is a synthetic dye commonly used in biological research and laboratory applications. It is a water-soluble, reddish-orange compound that can be used as a pH indicator, a staining agent, and a cell viability assay. The core function of neutral red is to provide a simple and reliable method for visualizing and quantifying living cells in various experimental settings.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (23 mentions) Seaplaque agarose, by Lonza (23 mentions) Dimethyl sulfoxide (dmso), by Merck Group (15 mentions) Penicillin, by Merck Group (12 mentions) Streptomycin, by Merck Group (11 mentions) Penicillin streptomycin, by Lonza (11 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions) Bovine serum albumin (bsa), by Merck Group (8 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions) Glacial acetic acid, by Merck Group (7 mentions) Phosphate buffered saline (pbs), by Merck Group (7 mentions) Gallic acid, by Merck Group (7 mentions) Fetal bovine serum (fbs), by Merck Group (6 mentions) Trypan blue, by Merck Group (6 mentions) Methanol, by Merck Group (6 mentions) Quercetin, by Merck Group (6 mentions) Triton x 100, by Merck Group (6 mentions) Trypsin edta, by Thermo Fisher Scientific (6 mentions) Ethanol, by Merck Group (5 mentions) Agarose, by Merck Group (5 mentions)

Close spelling variants of this product

Neutral red solution (84 citations) Neutral red dye (48 citations) Neutral red powder (5 citations) Neutral Red stain (4 citations) Neutral Red Assay (4 citations) Neutral red reagent (3 citations) Neutral Red Uptake Assay (3 citations) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), neutral red (NR) solution (3 citations) Neutral red uptake test (3 citations) Neutral red based in vitro toxicology assay kit (3 citations)

333 protocols using neutral red

1

Neutral Red Cytotoxicity Assay for Flavonoids

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Cells were plated in 96-well plates at 1 × 104 cells per well and allowed to adhere for 24 h. Cells were treated with flavonoids at concentrations of 3.1, 6.2, 12.5, 25, 50, and 100 μM and incubated for 24 or 48 h. Then neutral red uptake assay was performed according to protocol (Repetto et al., 2008 (link)). Briefly, the culture medium was replaced by the solution of neutral red (72210, Sigma-Aldrich) in medium at a concentration 40 μg/mL. Plates were incubated at 37 C for 2 h. The neutral red solution was then discarded, and neutral red absorbed by cells was dissolved in 50% ethanol containing 1% glacial acetic acid. Absorbance was measured at 540 nm using a microplate reader and IC50 values were calculated using GraphPad Prism software.
Szoka L., Nazaruk J., Giegiel J, & Isidorov V. (2023). Prolidase-proline oxidase axis is engaged in apoptosis induction by birch buds flavonol santin in endometrial adenocarcinoma cell line. Frontiers in Molecular Biosciences, 10, 1247536.
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2

Developmental and Adult Zebrafish Tissue Preparation

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4 dpi larvae were fixed in Dent’s Fixative (80% methanol, 20% DMSO), blocked in 3% goat serum and stained with primary and secondary antibodies.
For neutral red staining, 3dpf larvae were soaked in neutral red (Sigma N6264) 2.5µg/ml overnight at 28.5C.
2 wpi adult zebrafish were euthanized, fixed in 4% PFA and frozen in Neg-50 (Richard Allen Scientific). Zebrafish were cryosectioned at 20 µm.
For immunostaining, sections from adult fish, were blocked in 3% goat serum and stained with primary and secondary antibodies as indicated.
Cronan M.R., Beerman R.W., Rosenberg A.F., Saelens J.W., Johnson M.G., Oehlers S.H., Sisk D.M., Jurcic Smith K.L., Medvitz N.A., Miller S.E., Trinh L.A., Fraser S.E., Madden J.F., Turner J., Stout J.E., Lee S, & Tobin D.M. (2016). Macrophage Epithelial Reprogramming Underlies Mycobacterial Granuloma Formation and Promotes Infection. Immunity, 45(4), 861-876.
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3

Measuring Cell Viability under Oxidative Stress

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Cell viability tests under oxidative stress were conducted as previously described (Someya et al., 2009 (link); Han et al., 2016b (link)). The Txn2 knockdown HEI-OC1 cells were replated on a 96 well plate (3×104 cells per well) and treated with H2O2 at 0, 0.5, 0.75, 1, 1.5, 2, and 2.5 mM for 3 h. After 21 h, H2O2 solutions were replaced with medium containing 50 μg/ml neutral red (Sigma-Aldrich) and incubated for 3 h. After washing with PBS, the neutral red solubilizing solution composed of 50% ethanol, 49% water, and 1% glacial acetic acid was added to each well. The absorbance was read at 540 nm in the Synergy HTX microplate reader (BioTek).
Kim M.J., Han C., White K., Park H.J., Ding D., Boyd K., Rothenberger C., Bose U., Carmichael P., Linser P.J., Tanokura M., Salvi R, & Someya S. (2020). Txn2 haplodeficiency does not affect cochlear antioxidant defenses or accelerate the progression of cochlear cell loss or hearing loss across the lifespan. Experimental gerontology, 141, 111078.
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4

Vital Staining and Apoptosis Detection in Zebrafish

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For Neutral Red staining, zebrafish larvae collected at 60 hpf and 3 dpf were soaked in Neutral Red (Sigma-Aldrich, N6264; 2.5 mg/mL) overnight at 28.5 °C. For AO staining, embryos recovered at 22 hpf were incubated in 10 μg/mL AO stain (Sigma-Aldrich) dissolved in E3 medium in the dark for ~30 min. TUNEL staining was performed as previously described [62 (link)]. pH3 and immunofluorescence staining were carried out using the following antibodies: PDCD11 (Sigma-Aldrich; HPA017924; 1:100); pH3 (Santa Cruz Biotechnology; 1:500), TGF-β1 (Sigma-Aldrich, T0438; 25 μg/mL)
Yang R., Zhan M., Guo M., Yuan H., Wang Y., Zhang Y., Zhang W., Chen S., de The H., Chen Z., Zhou J, & Zhu J. (2020). Yolk sac-derived Pdcd11-positive cells modulate zebrafish microglia differentiation through the NF-κB-Tgfβ1 pathway. Cell Death and Differentiation, 28(1), 170-183.
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5

Staining Embryos with Neutral Red and Sudan Black

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For Neutral Red staining, embryos were collected and incubated in egg water with 2.5 μg/mL Neutral Red (Sigma-Aldrich) from 80 to 90 hpf [56 (link)]. For Sudan Black staining, fixed embryos were treated with a Sudan Black (Sigma-Aldrich) solution, as previously described [32 (link)]. Staining was then observed under a microscope.
Dong Z.W., Ren C.G., Xia Y., Su D., Du T.T., Fan H.B., Yuan H., Wang L., Dong M., Li W.C., Jin Y., Chen Y., Deng M., Liu T.X., Gu A.H, & Zhou Y. (2014). Pten regulates homeostasis and inflammation-induced migration of myelocytes in zebrafish. Journal of Hematology & Oncology, 7, 17.
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6

Visualizing Drosophila Egg Follicle Development

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Collected D. melanogaster laid -transgenic- follicles were stained with 0.06% 3-3′-diaminobenzidine (DAB) in 50 mM Tris-HCl, pH 7.6 and examined under visible light with a BMS stereomicroscope. Propidium iodide (PI) staining of follicles and embryos was carried out as previously described15 (link)16 . Transgenic (s36-targeted) follicles stained with PI or over-expressing the eGFP fluorescent protein were visualized under a Nikon confocal laser scanning microscope (CLSM), model Digital Eclipse C1 (Nikon, Tokyo, Japan). For the neutral-red staining assay, de-chorionated follicles were incubated for 10 min at room temperature with a solution of 5 mg/ml neutral red (Sigma-Aldrich, Missouri, USA) in 1x PBS, subsequently washed five times with 1x PBS containing 0.05% Triton X-100 and finally visualized under a BMS stereomicroscope. LM imaging was repeated three times with independent fly crosses for each experimental condition studied.
Velentzas A.D., Velentzas P.D., Sagioglou N.E., Konstantakou E.G., Anagnostopoulos A.K., Tsioka M.M., Mpakou V.E., Kollia Z., Consoulas C., Margaritis L.H., Papassideri I.S., Tsangaris G.T., Sarantopoulou E., Cefalas A.C, & Stravopodis D.J. (2016). Targeted Downregulation of s36 Protein Unearths its Cardinal Role in Chorion Biogenesis and Architecture during Drosophila melanogaster Oogenesis. Scientific Reports, 6, 35511.
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7

Neutral Red-Based Cell Growth Assay

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After transfection, cells were incubated for 5 d. The Idh2 knockdown cells were re-plated on a 12-well plate. Cells were allowed to grow from 24 to 96 h in DMEM. Cell growth rates were determined by the neutral red assay as previously described (Repetto et al., 2008). At completion of incubation, cells were incubated in DMEM with 50 µg/mL of neutral red (Sigma-Aldrich, St. Louis, MO) at 37 °C for 2–3 h. After washing, cells were then treated with 200 μl of neutral red solubilization solution (50% ethanol, 49% deionized water, and 1% glacial acetic acid (Sigma-Aldrich, St. Louis, MO) per well. The 12-well plate was incubated at room temperature on a plate shaker overnight. The OD (optical density) values of the neutral red extract in each well were measured at 540 nm in a microplate reader (Bio-Tek).
White K., Kim M.J., Han C., Park H.J., Ding D., Boyd K., Walker L., Linser P., Meneses Z., Slade C., Hirst J., Santostefano K., Terada N., Miyakawa T., Tanokura M., Salvi R, & Someya S. (2018). Loss of IDH2 Accelerates Age-related Hearing Loss in Male Mice. Scientific Reports, 8, 5039.
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8

Evaluating FTY720 Analogs' Cytoprotection

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FTY720s without H2O2 - Untransfected OLN-93 cells were seeded in 96-well plates at a density of 9×103 cells/well for 24 hr or 6×103 cells/well for 48 hr. After attachment and growth overnight, cells were treated with 40, 80 or 160 or 320 nM of FTY720, FTY720-C2, FTY720-Mitoxy or vehicle. After 24 or 48 hr, growth medium with FTY720s was removed and viability was measured by neutral red assay (Riedel et al., 2011 (link)), with minor modifications. After FTY720s, cells were washed with phosphate buffered saline (PBS) then incubated in growth medium containing 0.005% neutral red (Sigma-Aldrich, St. Louis, MO, Cat N664-50ML) for 2 hr. After incubation, cells were washed with PBS then treated with acidified ethanol (50% EtOH, 1% acetic acid), to release neutral red from cells. Absorbance was measured using a Multiskan Spectrum plate reader (Thermo Fisher Scientific, Waltham, MA) at λ 540 nm.
FTY720s ± H2O2 - Untransfected OLN-93 cells were plated in 96-well plates 2×103 cells/well. After attachment and overnight growth, cells were treated with 160 nM of FTY720, FTY720-C2, FTY720-Mitoxy or vehicle for 48 hr. Oxidative stress wells had 75 μM H2O2 or 100 μM H2O2 added for 2 hr. Positive controls received H2O2 + catalase (Sigma-Aldrich, St. Louis, MO, Cat C40, 800 U/well) for 2 hr; and negative controls had 70% methanol (100 μl) added 30 min prior to neutral red assay as detailed above.
Vargas-Medrano J., Segura-Ulate I., Yang B., Chinnasamy R., Arterburn J.B, & Perez R.G. (2019). FTY720-Mitoxy Reduces Toxicity Associated with α-Synuclein and Oxidative Stress by Increasing Trophic Factor Expression and Myelin Protein in OLN-93 Oligodendroglia Cell Cultures. Neuropharmacology, 158, 107701.
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9

Lysosomal Membrane Stability in Mussel Hemocytes

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The effect of PS-MP and PS-NP on the lysosomal membrane stability (LMS) of mussel hemocytes was evaluated using the Neutral Red Retention Assay (NRRA) as described by Martinez-Gomez et al. [28 ]. The method is based on exposing hemocytes to a solution containing 0.01% Neutral Red (NR, Sigma-Aldrich ®, Milan, Italy) acidophile dye (which migrates into lysosomes) and on the assessment of the Neutral Red Retention Time (NRRT), which is the time (min) where more than 50% of the lysosomes released the dye into the cytosol.
Capolupo M., Valbonesi P, & Fabbri E. (2021). A Comparative Assessment of the Chronic Effects of Micro- and Nano-Plastics on the Physiology of the Mediterranean Mussel Mytilus galloprovincialis. Nanomaterials, 11(3), 649.
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10

Cytotoxicity of Rare Honey Varieties

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We ran this assay, as this type of honey is quite rare and its safety profile poorly known. In vitro cytotoxicity was determined as described by Babich and Borenfreund (19 (link)). Briefly, CAL 27, HepG2, and Caco-2 cells were seeded in 96-well plates (105 cells per mL) and treated with STH, STHE, HGA, and AFH samples in four concentrations (Table 1) for 30 min, 1 h, and 2 h. After treatment, we added Neutral Red (Sigma-Aldrich) solution, incubated the cells at 37 °C for 1 h, removed Neutral Red solution, and washed the cells with phosphate buffered saline (PBS) (100 μL). Finally, we added 100 μL of solution combining acetic acid (Sigma-Aldrich), ethanol (Kemika), and demineralised water in the 0.1:5:4.9 ratio to extract the dye from the lysosomes of living cells. Cell viability was measured as colour intensity and absorption at 540 nm in a FLUOstar OPTIMA plate reader (BMG Labtech, Durham, NC, USA).
Data are expressed as percentage of viability of untreated cells (considered to be 100 %). Each concentration was tested in quadruplicate, and each experiment was repeated three times.
Jurič A., Huđek Turković A., Brčić Karačonji I., Prđun S., Bubalo D, & Durgo K. (2022). Cytotoxic Activity of Strawberry Tree (Arbutus Unedo L.) Honey, its Extract, and Homogentisic Acid on CAL 27, HepG2, and Caco-2 Cell Lines. Archives of Industrial Hygiene and Toxicology, 73(2), 158-168.
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