Dulbecco s phosphate buffered saline

Manufactured by Corning

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Dulbecco's phosphate-buffered saline (DPBS) is a balanced salt solution commonly used in cell culture and biological research. It is a sterile, isotonic buffer that maintains the pH and osmotic balance of cell samples. DPBS is formulated to preserve the viability and integrity of cells during various experimental procedures.

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Dulbecco’s phosphate-buffered saline (DPBS; (78 citations) Dulbecco’s phosphate buffered saline (PBS, (12 citations) Dulbecco’s Phosphate (2 citations)

35 protocols using dulbecco s phosphate buffered saline

Murine Ovarian Cancer Cell Lines

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DMEM (Dulbecco’s Modification of Eagle’s Medium), RPMI-1640, Hanks Balanced Salt Solution (HBSS), and Dulbecco’s Phosphate Buffered Saline (DPBS) from Cellgro (Manassas, VA) were the primary tissue culture media. SuperSignal West Dura Extended Duration Substrate was used for detection of protein bands on Western blots. RIPA buffer and Protease Inhibitor Cocktail were from ThermoFisher Scientific (Waltham, MA). All primary and secondary antibodies were from Cell Signaling Technology (Beverly, MA) or Jackson ImmunoResearch Laboratories (West Grove, PA), respectively. FITC-Annexin V Apoptosis Detection kit was purchased from BD Pharmingen (San Diego, CA). OVCAR3, ECC1, SKOV3, MCF7 and 4T1 cell lines were purchased from ATCC. The MYC-HRAS MOSE murine ovarian cancer cell line was a gift from Dr. Christine Walsh and was generated by introducing mutant c-myc and H-Ras genes into normal murine ovarian epithelial cells^{44 (link)}.

Kapur A., Beres T., Rathi K., Nayak A.P., Czarnecki A., Felder M., Gillette A., Ericksen S.S., Sampene E., Skala M.C., Barroilhet L, & Patankar M.S. (2018). Oxidative stress via inhibition of the mitochondrial electron transport and Nrf-2-mediated anti-oxidative response regulate the cytotoxic activity of plumbagin. Scientific Reports, 8, 1073.

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Culturing Primary Human vSMC

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Primary human vSMC from ScienCell (Catalog #1100, Carlsbad, California) were cultured under standard conditions (5% CO₂ at 37 °C) in Medium 231 containing 5% Smooth Muscle Growth Supplement (Gibco), 0.1 mg/mL Primocin (Invivogen, San Diego, California), and 100 U/mL each penicillin and streptomycin (Life Technologies, Thermo Fisher Scientific) in BioCoat Collagen I culture flasks (Corning, New York). Cells were passaged using 0.05% Trypsin EDTA (Gibco) after washing with sterile Dulbecco’s Phosphate Buffered Saline (Cellgro, Mediatech, Corning). For flow cytometry and RT-qPCR experiments, vSMC were plated in BioCoat Collagen I 96-well plates (Corning) at 1×10⁴ cells/well. After two days in growth-supplemented media, the cells were transitioned to Smooth Muscle Differentiation medium (Gibco).

Cupino T.L., Watson B.A., Cupino A.C., Oda K., Ghamsary M.G., Soriano S, & Kirsch W.M. (2017). Stability and Bioactivity of Chitosan as a Transfection Agent in Primary Human Cell Cultures: A Case for Chitosan-only Controls: Chitosan Modulates CD59 Expression. Carbohydrate polymers, 180, 376-384.

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Preparation of HOCl-Oxidized Tumor Cell Lysates

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We followed the protocol from a previous report to prepare HOCl-oxidized LL2 and EG7-OVA lysate [18 (link)]. Briefly, NaOCl reagent (Sigma-Aldrich) was diluted with DPBS (Dulbecco’s Phosphate Buffered Saline, Cellgro) and immediately added to the tumor cells to prepare a 0.7 μM HOCl solution. The final cell density was 1 × 10⁶ cells/ml in DPBS. The tumor cell suspensions were incubated at 37 °C for 1 h with gentle agitation every 30 min to induce oxidation-dependent tumor cell death. At the end of the 1 h treatment, the tumor cells were harvested, washed twice with DPBS (3 times the volume of DPBS was added to each volume of HOCl solution to ensure complete removal of HOCl), and subjected to 7 freeze-thaw cycles. For this, the TCLs were frozen in liquid nitrogen for ≥5 min and thawed completely at room temperature 7 times to completely rupture the tumor cells.

Zhang R., Tang L., Li Q., Tian Y., Zhao B., Zhou B, & Yang L. (2021). Cholesterol modified DP7 and pantothenic acid induce dendritic cell homing to enhance the efficacy of dendritic cell vaccines. Molecular Biomedicine, 2, 37.

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Isolation of Human CD14+ Monocytes

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Human white blood cells were collected from leukoreduction filters (Sepacell RS-2000; Fenwal Inc.) after processing of healthy volunteer donor whole blood (American Red Cross) after rinsing with 50 mL of selection buffer (2 mmol/L ethylenediaminetetraacetic acid (EDTA; J.T. Baker Inc.), 2% heat-inactivated fetal bovine serum (Sigma-Aldrich) in Dulbecco’s phosphate- buffered saline (Corning Cellgro)). The filtrate was layered onto Ficoll-Paque PLUS (GE Healthcare) to isolate mononuclear cells by density gradient centrifugation. Mononuclear cells treated with CD14-positive selection beads (BD Biosciences) in selection buffer and subsequent EasySep magnetic purification procedure (Stemcell Technologies) resulted in purified CD14+ monocytes (mean purity 95%), which were cryopreserved in Cryostor CS5 (Biolife Solutions). These primary human monocytes were labelled with BCECF-AM and used in adhesion assays as described above for THP-1 cells.

Rajendran N.K., Liu W., Chu C.C., Cahill P.A, & Redmond E.M. (2021). Moderate dose alcohol protects against serum amyloid protein A1-induced endothelial dysfunction via both notch-dependent and -independent pathways. Alcoholism, clinical and experimental research.

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Mammary Fibroblast Cell Culture

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DMEM (Dulbecco’s Modification of Eagle’s Medium), RPMI-1640, Hanks Balanced Salt Solution (HBSS), and Dulbecco’s Phosphate Buffered Saline (DPBS) were from Cellgro (Manassas, VA). SuperSignal West Dura Extended Duration Substrate, RIPA buffer and Protease Inhibitor Cocktail were from ThermoFisher Scientific (Waltham, MA). All primary and secondary antibodies were from Cell Signaling Technology (Beverly, MA) and Jackson ImmunoResearch Laboratories (West Grove, PA), respectively. FITC-Annexin V Apoptosis Detection kit was purchased from BD Pharmingen (San Diego, CA). Cancer cell lines used in this study were purchased from ATCC. The immortalized human mammary fibroblast cell line was a kind gift from Dr. Andreas Friedl (UW-Madison).

Kapur A., Felder M., Fass L., Kaur J., Czarnecki A., Rathi K., Zeng S., Osowski K.K., Howell C., Xiong M.P., Whelan R.J, & Patankar M.S. (2016). Modulation of oxidative stress and subsequent induction of apoptosis and endoplasmic reticulum stress allows citral to decrease cancer cell proliferation. Scientific Reports, 6, 27530.

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Virus Purification from Cell Culture

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Vero cells were infected at an MOI of 0.01 for 24 hours and culture supernatants were collected and filtered using a 0.2-μm bottle top filter (Millipore, Etobicoke, Canada). The supernatant was then centrifuged at 30,100 g for 1h30 and the pellet was resuspended in Dulbecco’s phosphate-buffered saline (Corning cellgro, Manassas, VA). The purified virus was kept at −80 °C.

Bourgeois-Daigneault M.C., Roy D.G., Falls T., Twumasi-Boateng K., St-Germain L.E., Marguerie M., Garcia V., Selman M., Jennings V.A., Pettigrew J., Amos S., Diallo J.S., Nelson B, & Bell J.C. (2016). Oncolytic vesicular stomatitis virus expressing interferon-γ has enhanced therapeutic activity. Molecular Therapy Oncolytics, 3, 16001-.

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Virus Rescue and Purification Protocol

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Virus rescues were performed as previously described. Vero cells were infected with T7 polymerase-expressing vaccinia Copenhagen virus at an MOI of 3. Following a 2 h incubation, media was removed and cells were transfected with T7-driven plasmids encoding VSV N, P, and L genes as well as the VSV∆51-B2 backbone. Supernatants collected 48 h post-transfection were passed through a 0.22 μm filter (MillexGP, Carrigtwohill, Ireland) to remove vaccinia Copenhagen virus.
For expansion and purification of the viral preparations, Vero cells were infected at an MOI of 0.001 and culture supernatants were collected 24 h post-infection. Supernatants were then filtered through a 0.2 μm bottle top filter (Millipore, Etobicoke, Canada) and centrifuged at 30,100 g for 90 min. The supernatant was discarded and pelleted virus was resuspended in Dulbecco’s phosphate-buffered saline (Corning cellgro, Manassas, VA). Purified virus was kept at − 80°C.

Bastin D., Aitken A.S., Pelin A., Pikor L.A., Crupi M.J., Huh M.S., Bourgeois-Daigneault M.C., Bell J.C, & Ilkow C.S. (2018). Enhanced susceptibility of cancer cells to oncolytic rhabdo-virotherapy by expression of Nodamura virus protein B2 as a suppressor of RNA interference. Journal for Immunotherapy of Cancer, 6, 62.

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Shikonin Cytotoxicity and Signaling

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Cell culture medium, Dulbecoo’s modified Eagle’s medium (DMEM), DMEM/F12, alpha-Minimum essential medium, trypsin, penicillin–streptomycin, and Dulbecco’s Phosphate Buffered Saline (DPBS) were purchased from Corning Cellgro (Manassas, VA, USA). Fetal bovine serum (FBS) was purchased from Gibco (Invitrogen, Carlsbad, CA, USA). Purified shikonin (≥98%), dimethyl sulfoxide (DMSO), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), hydrochloric acid (HCl), isopropanol, RIPA buffer, protease inhibitor cocktail and Tris-buffered saline/Tween 20 (TBST) were purchased from Sigma (St. Louis, MO, USA). Antibodies against dual specificity phosphatase (DUSP)-1, DUSP-2, β-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against mouse phospho-JNK 1/2, JNK 1/2, phospho-p38 mitogen-activated protein kinase (MAPK), and p38 MAPK, and phospho-ERK 1/2, ERK 1/2 were purchased from Cell Signaling (Farmingdale, NY, USA). Pierce BCA Protein Assay Kit and ECL chemiluminescence substrate were purchased from Thermo Scientific (Rockford, IL, USA). TRIzol reagent, SuperScript® VILO™ cDNA Synthesis kit, SYBR® GreenER™ qPCR SuperMixes were purchased from Life Technologies (Carlsbad, CA, USA). RNA 6000 Nano LabChip kit was obtained from Agilent Technologies (Palo Alto, CA, USA).

Lin K.H., Huang M.Y., Cheng W.C., Wang S.C., Fang S.H., Tu H.P., Su C.C., Hung Y.L., Liu P.L., Chen C.S., Wang Y.T, & Li C.Y. (2018). RNA-seq transcriptome analysis of breast cancer cell lines under shikonin treatment. Scientific Reports, 8, 2672.

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Immunostaining of hESC-derived ONP Spheroids

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For hESC-derived late-stage ONP spheroids, culture medium was first removed and spheroids were fixed with 4% (w/v) paraformaldehyde for 30 minutes followed by blocking overnight in a solution composed of 5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) and 0.1% Triton X-100 in Dulbecco’s Phosphate-Buffered Saline (Corning Life Sciences, Tewksbury, MA, USA). Each sample was incubated for three days at room temperature with the primary antibodies listed in Supplementary Table S3. This was followed by several phosphate-buffered saline (PBS) washes and incubation in the dark for three days at room temperature with Alexa Fluor 488 and 647 (#ab150129, #ab150075; Abcam, Cambridge, MA, USA). Nuclei were stained with 4’ 6-diamidino-2-phenylindole (DAPI; #D1306; Thermo Fisher Scientific, Waltham, MA, USA) for 30 minutes. Images were then assessed using a Nikon A1 confocal microscope (Nikon, Tokyo, Japan). ImageJ ver. 2.0.0-rc-69/1.52p. (National Institutes of Health, Bethesda, MA, USA) was used to quantify the images. Further details on image acquisition and quantification of immuno-positive cells are described in the Supplementary Data.

Chang H.T., Heuer R.A., Oleksijew A.M., Coots K.S., Roque C.B., Nella K.T., McGuire T.L, & Matsuoka A.J. (2020). An Engineered Three-Dimensional Stem Cell Niche in the Inner Ear by Applying a Nanofibrillar Cellulose Hydrogel with a Sustained-Release Neurotrophic Factor Delivery System. Acta biomaterialia, 108, 111-127.

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Single-cell Whole Genome Sequencing

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We tested the whole genome of 20 HeLa-CCL2 cells. As control, 10 normal human leukocytes from the blood of a healthy volunteer were also sequenced. Add 3 volumes of red blood cell lysis buffer (RBC Lysis Buffer, CWBiotech) to the blood sample and put the sample on ice for 15 minutes. Then centrifuged the sample at 1200 rpm for 5 minutes to removed supernatant and add 1.0 ml DPBS (1X Dulbecco’s Phosphate Buffered Saline, without calcium and magnesium, Corning) to resuspend the leukocytes. Single cells were captured by mouth pipette under the stereomicroscope (Nikon SZM745). The single-cell DNA amplification was followed by the MALBAC technique developed by Xie’s laboratory [14 (link)]. After amplification, six genomic loci were checked in the samples by qPCR before generating library to make sure the DNA was well presented as quality control (QC). We picked the sample with over two detectable loci to generate library. The sequencing depth for a single cell was 0.3X (about 1G per cell).

Hu W.E., Zhang X., Guo Q.F., Yang J.W., Yang Y., Wei S.C, & Su X.D. (2019). HeLa-CCL2 cell heterogeneity studied by single-cell DNA and RNA sequencing. PLoS ONE, 14(12), e0225466.

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