Phenol red

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Germany, Japan, France, Australia, Netherlands, Italy, Macao

Phenol red is a pH indicator used in various laboratory applications. It is a weakly acidic dye that changes color in response to changes in pH. Phenol red exhibits a yellow color in acidic solutions and a pink/red color in alkaline solutions, allowing it to be used to monitor pH changes in biological and chemical systems.

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Phenol red-free M199 Leibowitz's L-15 medium without phenol red RPMI-1640- GlutaMAX-I without phenol red Phenol red and Hepes TrypLE Express enzyme with phenol red DMEM/F12 no phenol red Gibco 0.25% (w/v) trypsin/phenol red HBSS without phenol red Dulbecco’s modified Eagle’s medium without phenol red DMEM (no phenol red)

557 protocols using phenol red

Multicolor Staining for NK Cell Cytotoxicity Assay

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5 µM Calcein Red AM (AAT Bioquest, Catalog no. 21900) and 10 µM Cell Tracker Blue dyes (Invitrogen, C2110) were used to stain primary NK cells and target cells (K562 or Jurkat), respectively. For staining, around 2 million cells were washed with RPMI free of supplements and Phenol red (Gibco, Catalog no. 11835030), resuspended in the freshly prepared dye solution in-RPMI (1 mL) and incubated at 37 °C for 30 min. Thereafter, cells were subsequently washed twice and resuspended in RPMI medium supplemented with 25 mM HEPES (Gibco, Catalog no. 15630056), 2% human serum (Sanquin), without Phenol red) containing 2.5 µM Sytox green (Invitrogen, Catalog no. S7020) and 7 µM CellEvent Caspase-3/7 Green Detection Reagent (Invitrogen, Catalog no. C10423), respectively. The stained cells were then proceeded for cell encapsulation.

Subedi N., Van Eyndhoven L.C., Hokke A.M., Houben L., Van Turnhout M.C., Bouten C.V., Eyer K, & Tel J. (2021). An automated real-time microfluidic platform to probe single NK cell heterogeneity and cytotoxicity on-chip. Scientific Reports, 11, 17084.

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Culturing and Preparing MRMT1-Luc2 Rat Mammary Carcinoma Cells

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Syngeneic MRMT1-Luc2 rat mammary gland carcinoma cells (Leiden University medical Center, The Nederlands) were cultured in RPMI 1640 medium without phenol red (Invitrogen, Paisley, UK/Nærum, Denmark) supplemented with 10% heat-inactivated foetal bovine serum, 1% L-glutamine and 2% penicillin/streptomycin (Invitrogen, Paisley, UK/Nærum, Denmark). On the day of surgery, MRMT1-Luc2 carcinoma cells were released by brief exposure to 0.1% w/v trypsin-EDTA and centrifuged in medium for 3 min at 1200rpm. The pellet was washed twice with Hanks’ balanced salt solution (HBSS) without calcium, magnesium or phenol red (Invitrogen, Paisley, UK/Nærum, Denmark) and centrifuged for 3 min at 1200rpm. Cells were re-suspended in HBSS and kept on ice until use.

Falk S., Al-Dihaissy T., Mezzanotte L, & Heegaard A.M. (2015). Effect of sex in the MRMT-1 model of cancer-induced bone pain. F1000Research, 4, 445.

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Lung Cell Isolation and Flow Cytometry

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Lungs were harvested, cut into small pieces with scissors, and resuspended with 5 mg/mL collagenase and 1 mg/mL DNase I in 1 mL of RPMI 1640 without phenol red (Gibco). After incubation at 37°C for a half hour, the lung samples were meshed with the plunger of a syringe on a 70μm cell strainer and rinsed with RPMI 1640 without phenol red. To determine CFUs, 10 μL were removed and plated as described above.
To analyze the lung cells by flow cytometry, red blood cells (RBC) were lysed with RBC buffer (Invitrogen), and the remaining cells were washed and resuspended in 1 mL of RMPI 1640 without phenol red. Then, 100 μL of the sample were stained for cell surface antigens. First, Fc receptors were blocked with anti-mouse CD16/CD32 monoclonal antibody 2.4G2 (BD Pharmingen) following the manufacturer’s instruction. Surface markers were stained with antibodies listed in Table 2. Flow cytometer data were acquired with a 5-Laser Cytek Aurora cytometer and analyzed with FlowJo X software (Tree Star Inc.)

Lee C.K., Oliveira L.V., Akalin A., Specht C.A., Lourenco D., Gomez C.L., Ramirez-Ortiz Z.G., Wang J.P, & Levitz S.M. (2023). Dysregulated Pulmonary Inflammatory Responses Exacerbate the Outcome of Secondary Aspergillosis Following Influenza. bioRxiv.

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Cell Culture and Transfection Workflow

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COS-7 and CHO cells were maintained in Dulbecco’s Modified Eagle’s Medium containing 25 mM D-glucose, 1 mM sodium pyruvate and 4 mM L-glutamine (Invitrogen, Carlsbad, CA) with 10% supplemented Fetal Bovine Serum (FBS) (Sigma Aldrich, St. Lois, MO) in T25 flasks (37 °C and 5% CO₂). Cells were passaged at 95% confluency using 0.05% TrypLE with Phenol Red (Invitrogen) and seeded onto 24-well Multiwell Plates (Falcon, Corning, NY) at a dilution of 1:20. Cells were transiently transfected using Lipofectamine 2000 according to manufacturer’s protocols (Invitrogen). Post-transfection, cells were treated with 0.05% TrypLE with Phenol Red (Invitrogen) and plated in 96-well tissue culture plates (Sarstedt, Numbrecht, Germany) at a dilution of 1:4 for imaging.

Qudrat A, & Truong K. (2016). Modular assembly of synthetic proteins that span the plasma membrane in mammalian cells. BMC Biotechnology, 16, 88.

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MCF7 Breast Cancer Cell Assay

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The breast cancer cell line MCF7 was obtained from American Type Culture Collection (ATCC, Manassas, VA). The cells were plated and grown for 24 h in DMEM containing phenol red and supplemented with 10% serum, 2 mM l-glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin (all from Life Technologies GmbH). For hormone deprivation experiments, cells were grown for three days in DMEM without phenol red (Life Technologies GmbH) and supplemented with 5% charcoal stripped heat-inactivated FBS (HyClone), 2 mM l-glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin. At day three, cells were stimulated with vehicle (ethanol) or 100 nM estradiol (Sigma–Aldrich).

Fleischer T., Tekpli X., Mathelier A., Wang S., Nebdal D., Dhakal H.P., Sahlberg K.K., Schlichting E., Børresen-Dale A.L., Borgen E., Naume B., Eskeland R., Frigessi A., Tost J., Hurtado A, & Kristensen V.N. (2017). DNA methylation at enhancers identifies distinct breast cancer lineages. Nature Communications, 8, 1379.

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Isolation and Characterization of Human Polymorphonuclear Leukocytes

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Human peripheral blood polymorphonuclear leukocytes (PMNs) were isolated from healthy donors (in accordance with relevant national guidelines and regulations, and informed consent of healthy volunteers: GS1-EK-4/543–2018) using EasySep Direct Human Pan-Granulocyte Isolation Kit (Stemcell). Purity and non-activated status of PMNs were validated using flow cytometry (FSC, SSC, CD1 lb, CD 14, CD45, CD62L, CD66b, CD235a; Miltenyi). For short-term storage or staining, PMNs were kept in Hanks balanced salt solution without phenol red (Gibco), supplemented with 0.6% human serum albumin (Sigma) and 1% glutamine (Gibco), with gently agitation in a ventilated tube in a humidified incubator. For experiments, PMNs were resuspended in RPMI without phenol red, supplemented with 0.6% human serum albumin, 20 mM HEPES (Gibco) and 1% glutamine.

Renkawitz J., Kopf A., Stopp J., de Vries I., Driscoll M.K., Merrin J., Hauschild R., Welf E.S., Danuser G., Fiolka R, & Sixt M. (2019). Nuclear positioning facilitates amoeboid migration along the path of least resistance. Nature, 568(7753), 546-550.

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Paracellular Permeability Assessment

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At t = 48 h, a FD4 (Sigma-Aldrich) permeability assay was performed to assess paracellular permeability of the cell monolayer. First, the phenol red-containing medium was discarded, the cells were washed with PBS and the same culture medium without phenol red (Gibco) was added. Before the start of the assay, the cells were left in the incubator for 1 h to become stable again. A concentration of 1.6 mg/mL FD4 was added to the apical side of the IEC and 100 uL samples of the basolateral medium were taken 1, 4 or 24 h after the addition of the FD4 and collected in a white 96-well plate (Corning). The taken samples were measured at Ex/Em = 492/518 nm with Fluorskan Ascent FL (Thermo Labsystems, Waltham, MA, USA).

Korsten S.G., Vromans H., Garssen J, & Willemsen L.E. (2023). Butyrate Protects Barrier Integrity and Suppresses Immune Activation in a Caco-2/PBMC Co-Culture Model While HDAC Inhibition Mimics Butyrate in Restoring Cytokine-Induced Barrier Disruption. Nutrients, 15(12), 2760.

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Murine Peritoneal Macrophage Isolation

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PMM were obtained from Swiss male mice (18–20 g) previously stimulated with 3% thioglycolate as reported (Santos et al., 2022 (link)). Four days after stimulation, PMM were collected by rinsing the mice's peritoneum with 10 mL of RPMI 1640 with phenol red, the cells seeded in 24- (3 × 10⁵ cells well⁻¹) and 96- (5 × 10⁴ cells well⁻¹) well plates and maintained for infection and cytotoxicity assays, respectively. PMM cell cultures were sustained in a 37°C incubator with 5% CO₂ in RPMI 1640 medium (pH 7.2–7.4) with phenol red and without phenol red (Gibco BRL) for infection analysis and cytotoxicity assays, respectively, supplemented with 1% (v/v) L-glutamine 200 mm, 1% (v/v) penicillin–streptomycin (10 000 U mL⁻¹ to 10 mg mL⁻¹) and 10% (v/v) FBS (Santos et al., 2022 (link)).

Present C., Girão R.D., Lin C., Caljon G., Van Calenbergh S., Moreira O., Ruivo L.A., Batista M.M., Azevedo R., Batista D.D, & Soeiro M.D. (2024). N6-methyltubercidin gives sterile cure in a cutaneous Leishmania amazonensis mouse model. Parasitology, 151(5), 506-513.

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Cell Culture and Treatments Optimization

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LNCaP and C42B cells were cultured in Rosewell Park Memorial Instiute 1640 (RPMI 1640, GIBCO). PC3, HEK293T, and DU145 were cultured in Dulbecco’s Modified Eagles Medium (DMEM). NE-1–3 and H-660 were cultured in RPMI1640 without phenol red (GIBCO). All base mediums were supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 100 U/mL penicillin and 100 μg/mL streptomycin, in an atmosphere of 95% air and 5% CO2, except for NE-1–3 and H-660 that 5% Charcoal Stripped Serum (CSS) was used. Lentiviruses and retroviruses were prepared and used as previously described. The following treatments were applied as follows; rapamycin was used at 100 nM, cycloleucine was used at 2 mM with daily media change, decitabine was used at 5 μM, and DHT was used at 10 nM. Androgen Deprivation Therapy conditions consisted on RPMI1640 media without phenol red (GIBCO), 10% dialyzed FBS, Glutamax and 100 U/mL penicillin 100 and 100 μg/mL streptomycin.

Reina-Campos M., Linares J.F., Duran A., Cordes T., L’Hermitte A., Badur M.G., Bhangoo M.S., Thorson P.K., Richards A., Rooslid T., Garcia-Olmo D.C., Nam-Cha S.Y., Salinas-Sanchez A.S., Eng K., Beltran H., Scott D.A., Metallo C.M., Moscat J, & Diaz-Meco M.T. (2019). Increased Serine and One Carbon Pathway Metabolism by PKCλ/ι Deficiency Promotes Neuroendocrine Prostate Cancer. Cancer cell, 35(3), 385-400.e9.

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T-cell Calcium Influx Assay

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Thymocytes were loaded with Indo-1 (Thermo Fisher Scientific, Waltham, MA, USA) for 45 min at 37 °C in RPMI 1640 without phenol red (Gibco Thermo Fisher Scientific, Waltham, MA, USA) and washed with RPMI without phenol red for 45 min at 37 °C. Stimulation was induced by the addition of neutravidin cross-linked CD3ε (145-2C11; BioLegend, San Diego, CA, USA). As a positive control, 100 nM ionomycin (Sigma-Aldrich, Saint Louis, MO, USA) was added 8–10 min after Ab stimulation. Ca²⁺ influx was measured with a BD Fortessa II (BD Biosciences, Franklin Lakes, NJ, USA) using a 325 nm laser line of a helium cadmium laser. Emission wavelength ranges from 395 and 500 to 520 nm were detected, and the ratio of the two emission intensities was calculated and analyzed with FlowJo (BD Biosciences, Franklin Lakes, NJ, USA).

Kästle M., Merten C., Hartig R., Plaza-Sirvent C., Schmitz I., Bommhardt U., Schraven B, & Simeoni L. (2022). Type of PaperY192 within the SH2 Domain of Lck Regulates TCR Signaling Downstream of PLC-γ1 and Thymic Selection. International Journal of Molecular Sciences, 23(13), 7271.

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