MCF-7 breast cancer cells were seeded in 6-well tissue culture plates at a density of 5 × 105 cells/well and allowed to grow overnight. The next day, cells were treated with coibamide A (10 nM - 100nM), brefeldin A (5 µM) or vehicle (0.1% DMSO) for 18 hours, harvested with trypsin and rinsed in cold Hank’s Balanced Salt Solution (HBSS; Thermo Fisher Scientific) supplemented with 0.1 % BSA (Amresco, Albany, NY). For cell surface detection of HER2, cells were stained with PE anti-human erbB2/HER-2 mouse mAb (# 324406; BioLegend, San Diego, CA) or PE mouse IgG1, k isotype control (FC) mouse mAb (# 400113; BioLegend) for 30 min at 4°C, washed once and resuspended in HBSS/0.1% BSA solution. All cell samples were analyzed by flow cytometry using an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA) and flow cytometry data analyzed with FlowJo Software (FlowJo LLC, BD Biosciences).
Hanks balanced salt solution (hbss)
Manufactured by BioLegend
HBSS (Hank's Balanced Salt Solution) is a balanced salt solution commonly used in cell culture and biological research applications. It is a sterile, buffered solution that provides a physiologically balanced ionic environment to support the maintenance and growth of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by BioLegend (1 mentions)
Bovine serum albumin (bsa), by Avantor (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Foxp3 transcription factor staining buffer kit, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Hanks balanced salt solution (hbss), by Harvard Bioscience (1 mentions)
Ccl21, by BioLegend (1 mentions)
Anti cd3 145 2c11, by BD (1 mentions)
Cxcl12, by BioLegend (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
3 protocols using hanks balanced salt solution (hbss)
1
Probing HER2 Expression in MCF-7 Cells
2
Multiparameter Phenotyping of Immune Cells
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Surface proteins were stained for 10 min on ice in HBSS (catalog number: 21-021-CV) with the following antibodies: Vα2 (B20.1), CD8α (53-6.7), CD45.1 (A20), CD45.2 (104), KLRG1 (2F1/KLRG1), CD127 (A7R34), CD27 (LG.3A10), CX3CR1 (SA011F11), CD44 (IM7), CD62L (MEL-14), CD69 (H1.2F3), CD103 (2E7), all purchased from BioLegend. For intracellular protein staining, samples were fixed in 2% paraformaldehyde (catalog number: 15710; Electron Microscopy Services) at room temperature for 45 min. Cells were then permeabilized using the FoxP3/Transcription Factor Staining Buffer Kit (catalog number: 00-5523-00; Thermo Fisher Scientific) and stained for 8 h at 4°C with the following antibodies: Tcf7 (C63D9), Eomes (Dan11mag), Bcl2 (BCL/1064), Ezh2 (11/EZH2), Ki67 (B56), Zeb1 (E2G6Y), Lef1 (C12A5), Gzma (GzA-3G8.5), T-bet (4B10), H3K27me3 (C36B11), and K3K4me3 (C42D8), purchased from BioLegend, Cell Signaling, BD Biosciences, and Thermo Fisher Scientific.
3
T Cell Adhesion Assay
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MACS-purified splenic T cells were stimulated with anti-CD3 (145-2C11; BD Biosciences, Franklin Lakes, NJ, USA), PMA (50 ng/ml; Merck Millipore, Billerica, MA, USA), or MnCl 2 for 30 min at 37°C before adhesion on Fc-ICAM-1 (10 mg/ml; R&D Systems, Minneapolis, MN, USA)-coated 96-well strips. Nonbound cells were removed by washing with HBSS (Biochrom AG, Berlin, Germany), and the bound cell fraction was determined by counting (in triplicates). To assess CXCR4/CCR7-mediated adhesion, T cells were incubated for 10 min at 37°C on Fc-ICAM-1-coated 96-well strips, coimmobilized with or without CXCL12 (100 ng/ml) or CCL21 (500 ng/ml; both from BioLegend); subsequently, nonbound cells were removed by washing with HBSS, as described above. Adherent cells were calculated as percentage of input (2 3 10 5 cells).
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