Hanks balanced salt solution (hbss)

Manufactured by Euroclone

Sourced in Italy

HBSS (Hank's Balanced Salt Solution) is a commonly used buffer solution in cell culture and biological research. It is a balanced salt solution that maintains the osmotic and pH balance of cells and tissues in vitro. The solution contains a mixture of inorganic salts, such as sodium chloride, potassium chloride, and calcium chloride, as well as glucose and phenol red as a pH indicator.

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Lab products found in correlation

Ethylene diamine tetraacetic acid (edta), by Merck Group (3 mentions) Dithiothreitol (dtt), by Merck Group (3 mentions) L glutamine, by Euroclone (2 mentions) Matrigel, by BD (2 mentions) Fetal bovine serum (fbs), by Merck Group (2 mentions) Dnase 1, by Merck Group (2 mentions) Hepes, by Lonza (2 mentions) Collagenase type 4, by Merck Group (2 mentions) Neurobasal medium, by Thermo Fisher Scientific (2 mentions) B27 supplement, by Thermo Fisher Scientific (2 mentions) Dulbecco s pbs, by Thermo Fisher Scientific (1 mentions) Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions) Rosiglitazone, by Cayman Chemical (1 mentions) Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions) Dipyrromethene boron difluoride bodipy 493 503 dye, by Thermo Fisher Scientific (1 mentions) 4 6 diamidin 2 phenylindole dapi containing mounting medium, by Abcam (1 mentions) Amphotericin b solution, by Thermo Fisher Scientific (1 mentions) Capsaicin, by Cayman Chemical (1 mentions) Improm iitm reverse transcription system, by Promega (1 mentions) Fluo 8 am, by Abcam (1 mentions)

19 protocols using hanks balanced salt solution (hbss)

Paclitaxel-primed Mesenchymal Stem Cells

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Drug loading was performed according to a modification of a standardized operating procedure previously set up for MSCs derived from several tissues (bone marrow, adipose tissue and gingiva) [12 (link),13 (link),14 (link),15 (link)]. Briefly, 5 × 10⁵ Hu-OBNSCs were exposed to 2 μg/mL PTX for 24 h. Then, the neurosphere cells were washed twice in Hank’s solution (HBSS, Euroclone, Pero, Italy). Paclitaxel-primed cells (hu-OBs/PTX) were then seeded in a 25 cm² flask to release the drug. After 24 h, conditioned media (CM), (h-OBs/PTX CM) was collected.
To measure the amount of drug internalized, each set of paclitaxel-primed cells was washed two times with Hank’s solution (HBSS, Euroclone, Pero, Italy) and suspended in complete medium (1 × 10⁶ cells/mL). The cells were lysed by sonication (three cycles of 0.4 s pulse at 30% amplitude each) (Labsonic U Braun, Reichertshausen, Germany) and centrifuged at 2500× g for 10 min and the lysate collected. The conditioned media and lysates from both PTX primed cells (h-OBs/PTX/CM; h-OBs/PTX/LYS) were tested for their anti-proliferative activity on standard cancer cell line CFPAC-1 and U87MG cells. Conditioned media and lysates from un-primed h-OBs (h-OBs/CM; h-OBs/LYS) were used as negative controls

Marei H.E., Casalbore P., Althani A., Coccè V., Cenciarelli C., Alessandri G., Brini A.T., Parati E., Bondiolotti G, & Pessina A. (2019). Human Olfactory Bulb Neural Stem Cells (Hu-OBNSCs) Can Be Loaded with Paclitaxel and Used to Inhibit Glioblastoma Cell Growth. Pharmaceutics, 11(1), 45.

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Isolation of Duodenal Immune Cells

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Fresh duodenal biopsies were collected in calcium- and magnesium-free Hanks’ balanced salt solution (HBSS, Euroclone, Italy) and rapidly processed. Biopsy specimens were initially incubated in a HBSS solution containing dithiothreitol (DTT, 0.145 mg/ml, Sigma-Aldrich, St. Louis, MO) for 15 min to remove mucus and adherent bacteria. After extensive washing in HBSS, biopsies were incubated for 50 min with a HBSS solution containing 0.37 mg/ml EDTA (Sigma-Aldrich) at 37 °C. At the end, supernatant was collected for intraepithelial lymphocytes (IELs) analysis. After extensive washing in HBSS, biopsies were then digested in RPMI 1640 medium (Euroclone, Italy), containing 400 U/ml collagenase D (Roche, Milan, Italy) in a 5% CO₂ incubator at 37 °C for 1 h. After incubation, LP mononuclear cells (LPMCs) released from the tissue samples were passed through a 70 μm cell strainer and washed with complete 10% RPMI 1640 medium.

Paroni M., Magarotto A., Tartari S., Nizzoli G., Larghi P., Ercoli G., Gianelli U., Pagani M., Elli L., Abrignani S., Conte D., Geginat J, & Caprioli F. (2016). Uncontrolled IL-17 Production by Intraepithelial Lymphocytes in a Case of non-IPEX Autoimmune Enteropathy. Clinical and Translational Gastroenterology, 7(7), e182-.

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Tissue Sampling for Leiomyoma Analysis

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Samples of myometrial tissue and leiomyoma were excised from two fertile women submitted to hysterectomy. The patients were Caucasians (age: 49 years and 43) and the position of the leiomyomas was intramural: the former with a single mass of 10 cm, the latter with two masses of 4 cm and 2 cm. Both patients displayed good general condition; none of them had a history of myomectomy or uterine surgery, had received medical therapy or oral contraceptives in the previous three months, or had evidence of genital tract infection, endometriosis, or ovarian disease. Patients gave their informed consent and the permission of the Human Investigation Committee was granted (Ethics Committee of Marche Region, Prot. N 2015 0486OR).
All experiments were performed in accordance with relevant guidelines and regulations.
The samples, immediately after hysterectomy, were collected in Hanks’ Balanced Salt Solution (HBSS) (Euroclone, Milan, Italy) and transferred to the laboratory for washing them with Dulbecco’s PBS (Invitrogen, Life Technologies, Carlsbad, CA, USA) in order to remove excess of blood and cutting them into small pieces.
In the former patient (P1), four pieces were taken from the fibrotic tissue (P1-L) and other four from the healthy myometrium (P1-Ctr); in the latter patient (P2), four pieces were taken from the leiomyoma (P2-L) and other three from the healthy myometrium (P2-Ctr).

Giuliani A., Greco S., Pacilè S., Zannotti A., Delli Carpini G., Tromba G., Giannubilo S.R., Ciavattini A, & Ciarmela P. (2019). Advanced 3D Imaging of Uterine Leiomyoma’s Morphology by Propagation-based Phase-Contrast Microtomography. Scientific Reports, 9, 10580.

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Cell Culture and Molecular Analysis

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Dulbecco’s modified Eagle medium (DMEM) enriched with 4.5 g/L D-glucose, 110 mg/L sodium pyruvate and 862 mg/L L-alanyl-L-glutamine (GlutaMAX^TM), DMEM/F-12 (1:1) medium enriched with GlutaMAX^TM, fetal bovine serum (FBS), penicillin/streptomycin solution and amphotericin B solution were purchased from Gibco by Life Technologies (Thermo Fisher Scientific, Inc., Waltham, MA, United States). Capsaicin and rosiglitazone were purchased from Cayman Chemical (Ann Arbor, MI, United States). Dipyrromethene boron difluoride (BODIPY) 493/503 dye, TRIzol reagent, PureLink^TM RNA Mini Kit and Platinum^TM SYBR^TM Green qPCR SuperMix-UDG kit for real time PCR were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, United States). ImProm-II^TM Reverse Transcription System was purchased from Promega (Madison, WI, United States). Normal goat (NG) serum was purchased by Vector Laboratories (Burlingame, CA, United States). 4,6-diamidin-2-phenylindole (DAPI)-containing mounting medium and Fluo-8 AM were purchased from Abcam (Cambridge, MA, United States). Hank’s balanced salt solution (HBSS) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased from Euroclone S.p.A. (Pero, Italy). All other chemicals used in the experiment and not listed above were purchased from Sigma-Aldrich (Darmstadt, Germany).

Montanari T., Boschi F, & Colitti M. (2019). Comparison of the Effects of Browning-Inducing Capsaicin on Two Murine Adipocyte Models. Frontiers in Physiology, 10, 1380.

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Loading GinPaMSCs with Paclitaxel

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To load GinPaMSCs with PTX, the cells were primed with a high amount of drug according to a standardized procedure previously described [7 (link),15 (link),17 (link)]. Briefly, cultures were obtained by seeding 2 × 10⁴ cells/cm², and after 72 h, cells were exposed to 2 µg/mL PTX for 24 h. Then, after washing twice with phosphate buffered saline (PBS), the cell monolayer was trypsinized, washed in Hank’s solution (HBSS)( Euroclone, Pero, Italy), and the PTX-primed cells (GinPaMSCs/PTX) were seeded in a 25 cm² flask in DMEM HG with 10% FBS and 2 mM l-glutamine (Euroclone, Pero, Italy) to release the drug. After 48 h of incubation into conditioned media (CM), PTX-loaded GinPaMSCs (GinPaMSCs/PTX/CM) were collected and tested in vitro for their anti-proliferative activity on different tumor cell lines (see Section 2.7). In particular, human pancreatic adenocarcinoma cell line CFPAC-1 was used as a standard laboratory assay according to the method reported below. CM from untreated MSCs were used as control.

Coccè V., Franzè S., Brini A.T., Giannì A.B., Pascucci L., Ciusani E., Alessandri G., Farronato G., Cavicchini L., Sordi V., Paroni R., Dei Cas M., Cilurzo F, & Pessina A. (2019). In Vitro Anticancer Activity of Extracellular Vesicles (EVs) Secreted by Gingival Mesenchymal Stromal Cells Primed with Paclitaxel. Pharmaceutics, 11(2), 61.

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Drug Loading of Mesenchymal Stem Cells

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The drug loading of GinPa-MSCs was performed by priming the cells according to a standardized procedure that use high drug dosage as previously described^{9 (link), 13 (link)}. Briefly, subconfluent cultures (2 × 10⁴ cells/cm²) were exposed to 2 µg/ml PTX, or DXR, or GCB for 24 hours. Then, the cells were washed twice with PBS, trypsinized and washed twice in Hank’s solution (HBSS, Euroclone UK). Drug-primed cells (GinPa-MSCs/PTX, GinPa-MSCs/DXR, GinPa-MSCs/GCB) were then seeded in a 25 cm² flask in DMEM high glucose with 10% FBS and 2 mM L-glutamine (Euroclone, UK) to release the drug. After 48 hours, conditioned media (CM), (GinPa-MSCs/PTX CM, GinPa-MSCs/DXR CM GinPa-MSCs/GCB CM) were collected and tested in vitro for their anti-proliferative activity on SCC154 cells and CFPAC-1 cells (used as standard assay). Conditioned Media from unprimed MSCs were used as control. To determine the amount of drug internalized and not released, cells collected after the releasing phase were lysed by three sonication cycles of 0.4 second pulse cycle and 30% amplitude each (Labsonic U Braun). Lysates from PTX, DXR and GCB primed cells (GinPa-MSCs/PTX LYS, GinPa-MSCs/DXR LYS, GinPa-MSCs/GCB LYS) and unprimed GinPa-MSCs were tested for their anti-proliferative activity.

Coccè V., Farronato D., Brini A.T., Masia C., Giannì A.B., Piovani G., Sisto F., Alessandri G., Angiero F, & Pessina A. (2017). Drug Loaded Gingival Mesenchymal Stromal Cells (GinPa-MSCs) Inhibit In Vitro Proliferation of Oral Squamous Cell Carcinoma. Scientific Reports, 7, 9376.

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Mitochondrial Oxidative Stress in Chondrocytes

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OA chondrocyte were seeded in 12 well-plates (8 × 10⁴ cells/well) for 24~h in DMEM with 10% FCS, before replacement with 0.5% FBS used for the treatment procedure. Then, the cells were incubated in Hanks’ Balanced Salt Solution (HBSS) (Euroclone, Milan, Italy) and MitoSOX Red for 15 min at 37 °C in dark, to evaluate mitochondrial superoxide anion (·O₂−) production. MitoSOX was dissolved in dimethyl sulfoxide (DMSO) (Euroclone, Milan, Italy), at a final concentration of 5 µM. Cells were then harvested by trypsin and collected into cytometry tubes and centrifuged at 1500 rpm for 10 min. Afterwards, cells were dissolved in saline solution before being analyzed by flow cytometry. A density of 1 × 10⁴ cells per assay (a total of 10,000 events) were measured by flow cytometry and data were analyzed with CellQuest software (Version 4.0, Becton Dickinson, San Jose, CA, USA). Results were collected as median of fluorescence (AU) and represented the mean of three independent experiments.

Cheleschi S., Barbarino M., Gallo I., Tenti S., Bottaro M., Frati E., Giannotti S, & Fioravanti A. (2020). Hydrostatic Pressure Regulates Oxidative Stress through microRNA in Human Osteoarthritic Chondrocytes. International Journal of Molecular Sciences, 21(10), 3653.

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Colorectal Cancer Epithelial Cell Isolation

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Colorectal cancer specimens were obtained from IRCCS Policlinico Ospedale Maggiore, Milan, Italy, under the same permission number as in 2.2. Specimens’ mucosa underwent sequential washes with Hanks’ Balanced Salt Solution (HBSS, Euroclone) containing DTT (0.1 mmol·L⁻¹, Sigma‐Aldrich) and EDTA (1 mmol·L⁻¹, Sigma‐Aldrich) in the presence of penicillin/streptomycin and gentamicin. Epithelial cells were obtained after collecting the supernatants from washes with EDTA, centrifuging at 200× g for 5 min, and preserving the pellet. Purity was confirmed via flow cytometry.

Díaz‐Basabe A., Burrello C., Lattanzi G., Botti F., Carrara A., Cassinotti E., Caprioli F, & Facciotti F. (2021). Human intestinal and circulating invariant natural killer T cells are cytotoxic against colorectal cancer cells via the perforin–granzyme pathway. Molecular Oncology, 15(12), 3385-3403.

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Adoptive Transfer of Anti-SSTR CAR T Cells

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Sixty-six 4–6 weeks old NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) female mice (Charles River, Lecco, Italy) were used for adoptive T cell transfer experiments. Briefly, each mouse received a subcutaneous injection of 2×10⁶ Luc⁺-CM or Luc⁺-BON1 cells resuspended in a 1:1 mixture of HBSS (Euroclone, Milan, Italy) and Matrigel (BD Bioscience). When tumors became palpable, at approximately day 15, mice were randomized to receive 7×10⁶ anti-SSTR CAR T cells (n=11), 7×10⁶ UT T cells (n=11), or phosphate-buffered saline (PBS) (n=11) via tail vein injection. The number of CAR-transduced CD8⁺ T cells administered to each mouse was equalized based on ddPCR analysis of CAR vector-positive cells. Following treatment in each group, mice received three daily intraperitoneal administrations of recombinant human IL-2 (Miltenyi Biotec) at 220 000 IU in 500 µL of PBS. Tumor growth was monitored weekly by an observer blinded to treatment allocation via in vivo BLI using an IVIS Lumina SIII instrumentation (Perkin Elmer). The tumor growth rate was quantified as the ratio between total photon flux at each investigational time-point and baseline levels. All animals were euthanized after 4 weeks from treatment, and tumors, brain, pancreas and spleen were harvested, fixed in formalin and embedded in paraffin. We used the ARRIVE checklist when writing our report.19

Mandriani B., Pellè E., Mannavola F., Palazzo A., Marsano R.M., Ingravallo G., Cazzato G., Ramello M.C., Porta C., Strosberg J., Abate-Daga D, & Cives M. (2022). Development of anti-somatostatin receptors CAR T cells for treatment of neuroendocrine tumors. Journal for Immunotherapy of Cancer, 10(6), e004854.

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Isolation of Peripheral Blood and Intestinal Immune Cells

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Healthy donors’ peripheral blood buffy coats were obtained from San Matteo Hospital, Pavia, Italy, whereas intestinal specimens were obtained from IRCCS Policlinico Ospedale Maggiore, Milan, Italy. Institutional Review Board (Milan, Area B) approved the study with permission number 566_2015. All methodologies were under full compliance with the Declaration of Helsinki, and informed consent was obtained from all subjects.
Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation using Ficoll‐Paque as for standard protocol. Natural killer cells were isolated from PBMCs using the NK Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Human lamina propria mononuclear cells (LPMCs) were isolated as previously described [53 (link)]. Briefly, the dissected intestinal mucosa was depleted of mucus and epithelial cells in sequential washes with Hanks’ Balanced Salt Solution (HBSS, Euroclone, Pero, Italy) containing DTT (0.1 mmol·L⁻¹, Sigma‐Aldrich, St Louis, MO, USA) and EDTA (1 mmol·L⁻¹, Sigma‐Aldrich) and then digested with collagenase D (400 U·mL⁻¹, Worthington Biochemical Corporation, Lakewood, NJ, USA) for 5 h at 37 °C. LPMCs were then separated with a Percoll gradient and cultured in RPMI‐1640 medium containing 5% human serum (Sigma‐Aldrich), penicillin/streptomycin, gentamicin, and 100 U·mL⁻¹ IL‐2 (Proleukin).

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