Facs stain buffer, by BD - Product details

1

Quantifying DR5 Expression in Tumor Cells

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Example 1

DR5 cell surface expression was quantitated by fluorescence activated cell sorting (FACS) analysis. Briefly, FACS stain buffer (BD Pharmigen Catalog #554656) was used for staining and wash steps. Tumor cells (1×10⁵-5×10⁵) were stained with 0.25 μg of anti-human DR5-PE (eBioscience Catalog #12-9908-42) or isotype-PE control (eBioscience Catalog #12-4714-42) for 15 minutes at 4° C., protected from light. Cells were washed twice, resuspended in FACS stain buffer, and results were acquired by flow cytometry. FIG. 1A shows the peak shift with the anti-human DR5 antibody (lower panel) as opposed to the isotype control (upper panel). FIG. 1B shows that DR5 expression varies by cell line, with 293F cells expressing the most DR5 among this set of cell lines.

US09938347B2. Tumor necrosis factor (TNF) superfamily receptor IgM antibodies and uses thereof (2018-04-10). IGM Biosciences A/S [DK]. Inventors: Beatrice Tien-Yi Wang [US], Max Allen Schwarzer [US], Bruce Alan Keyt [US], Ramesh Baliga [US].

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2

Quantification of DR5 Cell Surface Expression

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Example 1

DR5 cell surface expression was quantitated by fluorescence activated cell sorting (FACS) analysis. Briefly, FACS stain buffer (BD Pharmigen Catalog #554656) was used for staining and wash steps. Tumor cells (1×10⁵-5×10⁵) were stained with 0.25 μg of anti-human DR5-PE (eBioscience Catalog #12-9908-42) or isotype-PE control (eBioscience Catalog #12-4714-42) for 15 minutes at 4° C., protected from light. Cells were washed twice, resuspended in FACS stain buffer, and results were acquired by flow cytometry. FIG. 1A shows the peak shift with the anti-human DR5 antibody (lower panel) as opposed to the isotype control (upper panel). FIG. 1B shows that DR5 expression varies by cell line, with 293F cells expressing the most DR5 among this set of cell lines.

US10689449B2. Multimeric death domain-containing receptor-5 (DR5) antibodies and uses thereof (2020-06-23). IGM Biosciences, Inc. [US]. Inventors: Beatrice Tien-Yi Wang [US], Max A. Schwarzer [US], Bruce Alan Keyt [US], Ramesh Baliga [US].

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3

DR5 Antibody Binding Affinity Assay

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Antibody affinity and avidity were measured using surface plasmon resonance (SPR) and DR5 specificity by ELISA (Supplementary Methods). Colo205 and H-EMC-SS (50,000 cells/well) were incubated with IGM-8444 or anti-DR5 IgG for 35 minutes at 4°C. IgG and IgM isotype controls were used at 10 μg/mL. Cells were washed with FACS stain buffer (BD Biosciences), incubated with 1 μg/mL of goat anti-human kappa-Alexa Fluor 647 (Southern Biotech #2060–31) for 30 minutes at 4°C, washed, resuspended with 100 μL/well of 7-AAD (BD Biosciences) diluted in FACS stain buffer and analyzed using a Beckman Coulter CytoFLEX flow cytometer.

Wang B.T., Kothambawala T., Wang L., Matthew T.J., Calhoun S.E., Saini A.K., Kotturi M.F., Hernandez G., Humke E.W., Peterson M.S., Sinclair A.M, & Keyt B.A. (2021). Multimeric Anti-DR5 IgM Agonist Antibody IGM-8444 Is a Potent Inducer of Cancer Cell Apoptosis and Synergizes with Chemotherapy and BCL-2 Inhibitor ABT-199. Molecular Cancer Therapeutics, 20(12), 2483-2494.

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4

Maternal Phagocytic Function in Pregnancy

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Whole blood samples were collected from pregnant C57BL/6 dams (mated with BALB/c males) at 10.5 or 16.5 dpc (n = 6 each). Whole maternal blood (50 μL) was then incubated with 10 μL of GFP+ placenta-derived particles or 10 μL of pHrodo™ Green E. coli BioParticles (Life Technologies) for 15 min at 37°C or on ice. After incubation, the cells were washed with FACS stain buffer (BD Biosciences) and centrifuged at 400 x g for 5 min. The cells were then incubated with anti-mouse CD11b Alexa Fluor594, anti-mouse F4/80 APC, and anti-mouse Ly6G APC-Cy7 antibodies (Table S1) in FACS staining buffer (BD Biosciences) for 30 minutes at 4°C in the dark. After incubation, erythrocytes were lysed using Ammonium-Chloride-Potassium (ACK) lysing buffer (Lonza, Walkersville, MD), and the resulting leukocytes were collected after centrifugation at 400 x g for 5 min. Finally, the cells were washed and resuspended in 500 μL of FACS staining buffer and acquired using the BD LSRFortessa flow cytometer and FACSDiva 9 software. The analysis and figures were performed using FlowJo software version 10. The percentage of active phagocytic cells was calculated as the percentage of phagocytic cells at 37°C minus the percentage of phagocytic cells on ice.

Arenas-Hernandez M., Romero R., Gershater M., Tao L., Xu Y., Garcia-Flores V., Pusod E., Miller D., Galaz J., Motomura K., Schwenkel G., Para R, & Gomez-Lopez N. (2021). Specific Innate Immune Cells Uptake Fetal Antigen and Display Homeostatic Phenotypes in the Maternal Circulation. Journal of leukocyte biology, 111(3), 519-538.

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5

Tumor Cell Isolation and Characterization

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The tumors were dissected and tumor cells were isolated using gentleMACS dissociator (Miltenyi Biotech #130-096-730). In brief, the tumors were washed in PBS, cut into pieces using a scalpel and dispersed in dissociation mix. The tumor suspensions were transferred into gentleMACS C tubes (Miltenyi Biotech #130-096-334) shaken at 37°C and 100 rpm for 45 min using a gentleMACS dissociator. Cell suspensions were centrifuged at 400 × g and 4°C for 10 min, then re-suspended in PBS + 2% heat inactivated (hi) FCS. The tumor homogenates were filtered using a cell strainer (70 µm) and subsequently centrifuged at 400 × g and 4°C for 10 min. The cell pellets were incubated for 2 min on ice in 1 ml ACK lysis buffer (Gibco #A10492-01) and washed once with 10 ml PBS+2% hi FCS. The number of cells was determined using a Vi-Cell XR Cell viability analyzer (Beckmann Coulter) and GFP-expressing tumor cells were quantified after staining with the mouse CD45-BV421 (Biolegend, #30-F11) to exclude mouse immune cells and fixable viability dye eFl780 (eBioscience, #65-0865-14) to exclude dead cells. After 30 min of incubation at 4°C, cells were washed, resuspended in FACS stain buffer (BD Biosciences, # 554656), and analyzed (2 × 10⁵ gated on living cells).

Schlager S., Salomon C., Olt S., Albrecht C., Ebert A., Bergner O., Wachter J., Trapani F., Gerlach D., Voss T., Traunbauer A., Jude J., Hinterndorfer M., Minnich M., Schweifer N., Blake S.M., Zinzalla V., Drobits B., McConnell D.B., Kraut N., Pearson M., Zuber J, & Koegl M. (2020). Inducible knock-out of BCL6 in lymphoma cells results in tumor stasis. Oncotarget, 11(9), 875-890.

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6

Flow Cytometry Gastric DC Analysis

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For flow cytometry, cells were labeled with pre-determined optimum concentrations of antibodies at 4°C for 15 min, followed by washing in FACS Stain Buffer (BD Biosciences). For intracellular staining, cells were fixed and permeabilized with Cytofix/Cytoperm (BD Bioscience), and antibodies we added in the presence of BD PermWash buffer. Dead cells were labeled with LIVE/DEAD® yellow dye (Life Technologies, Carlsbad, CA). A BD LSR or LSRII was used for flow cytometry; and data were analyzed using FlowJo V10 software (Treestar, Ashland, OR). Gastric DCs were gated as live CD45^pos/lineage^neg/HLA-DR^high cells. The lineage cocktail contained antibodies to CD3, CD19, and CD56.

Swain S., Roe M.M., Sebrell T.A., Sidar B., Dankoff J., VanAusdol R., Smythies L.E., Smith P.D, & Bimczok D. (2018). CD103 (αE Integrin) Undergoes Endosomal Trafficking in Human Dendritic Cells, but Does Not Mediate Epithelial Adhesion. Frontiers in Immunology, 9, 2989.

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7

Isolation of CD31+ endothelial cells

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We obtained CD31⁺ endothelial cells by cell sorting, essentially as previously reported [14 (link)]. Unspecific binding was blocked by incubation for 10 min with anti CD16/CD32 (Fc block, clone 2.4G2; #553142, BD Pharmingen, RRID:AB_394657) in FACS buffer at 4 °C. Live/dead Aqua Dead Cell stain kit (#L34957, Thermo Fisher Scientific) was used to determine the viability of cells. Cells were incubated with the following primary antibodies during 30 min at 4 °C: CD11b (clone M1/70, APC-Cy7, #557657, BD Pharmingen, RRID:AB_396772), CD45 (clone 30-F11, FITC, #553080, BD Pharmingen, RRID:AB_394610), CD31 (clone 390, PE-Cyanine7, #25-0311-82, eBioscience, RRID:AB_2716949). After washing with FACS Stain Buffer (#554656, BD Biosciences), the cells were sorted in a FACSAriaII or FACSAria SORP sorter (BD Biosciences). Endothelial cells were collected in sterile DPBS (#14190-094, Thermo Fisher Scientific), centrifuged, and resuspended in lysis buffer (from PureLink™ RNA Micro Kit #12183016, Invitrogen) supplemented with 10% β-mercaptoethanol and finally snap-frozen in dry ice.

Arbaizar-Rovirosa M., Gallizioli M., Lozano J.J., Sidorova J., Pedragosa J., Figuerola S., Chaparro-Cabanillas N., Boya P., Graupera M., Claret M., Urra X, & Planas A.M. (2023). Transcriptomics and translatomics identify a robust inflammatory gene signature in brain endothelial cells after ischemic stroke. Journal of Neuroinflammation, 20, 207.

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8

Analyzing Kupffer Cell Polarization

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Kupffer cells (1 × 10⁵ cells/mL) were mixed with 100 µL FACS stain buffer (BD Bioscience, San Jose, CA, USA), followed by staining with CD11c (M1 macrophage marker) and CD206 (M2 macrophage marker) for 30 min under darkness to analyze Kupffer cell polarization. Samples were resuspended with the stain buffer, and then were analyzed using the FlowJo software package (Tree Star, Ashland, OR, USA).

Yao J, & Zhao Y. (2023). Lp-PLA2 silencing ameliorates inflammation and autophagy in nonalcoholic steatohepatitis through inhibiting the JAK2/STAT3 pathway. PeerJ, 11, e15639.

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9

Isolation of CD31+ endothelial cells

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We obtained CD31⁺ endothelial cells by cell sorting, essentially as previously reported [14 (link)]. Unspecific binding was blocked by incubation for 10 min with anti CD16/CD32 (Fc block, clone 2.4G2; #553142, BD Pharmingen, RRID:AB_394657) in FACS buffer at 4 °C. Live/dead Aqua Dead Cell stain kit (#L34957, Thermo Fisher Scientific) was used to determine the viability of cells. Cells were incubated with the following primary antibodies during 30 min at 4 °C: CD11b (clone M1/70, APC-Cy7, #557657, BD Pharmingen, RRID:AB_396772), CD45 (clone 30-F11, FITC, #553080, BD Pharmingen, RRID:AB_394610), CD31 (clone 390, PE-Cyanine7, #25-0311-82, eBioscience, RRID:AB_2716949). After washing with FACS Stain Buffer (#554656, BD Biosciences), the cells were sorted in a FACSAriaII or FACSAria SORP sorter (BD Biosciences). Endothelial cells were collected in sterile DPBS (#14190-094, Thermo Fisher Scientific), centrifuged, and resuspended in lysis buffer (from PureLink™ RNA Micro Kit #12183016, Invitrogen) supplemented with 10% β-mercaptoethanol and finally snap-frozen in dry ice.

Arbaizar-Rovirosa M., Gallizioli M., Lozano J.J., Sidorova J., Pedragosa J., Figuerola S., Chaparro-Cabanillas N., Boya P., Graupera M., Claret M., Urra X, & Planas A.M. (2023). Transcriptomics and translatomics identify a robust inflammatory gene signature in brain endothelial cells after ischemic stroke. Journal of Neuroinflammation, 20, 207.

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10

Flow Cytometric Analysis of Proliferated T Cells

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The following fluorochrome-conjugated antibodies (mAb) were used. For the in vitro assay, Dead Cell Marker-FarRed (Life Technologies), CD3-DAPI (Biolegend, San Diego, CA, USA), CD45-BV510 (Biolegend), CD4-FITC (eBiosciences, Thermo Fisher Scientific, Waltham, MA, USA), CD8-PE (eBiosciences), CD11c-PE-Cy7 (eBiosciences), and CellTrace Far Red-APC (Invitrogen, Waltham, MA, USA) were used. For the in vivo studies, Dead Cell Marker-Aqua (Life Technologies), CD3-DAPI (Biolegend), CD45.2-PE (eBiosciences), CD45.1-eFluor780 (eBiosciences), CD4-FITC (eBiosciences), CD8-BV421 (BD), CD11c-PE-Cy7 (eBiosciences), CD25-PE Cy7 (eBiosciences), CD19-BV711 (BD) and CellTrace Far Red-APC (Invitrogen) were used. For cell surface analysis of proliferation, CD4+ T cells were collected after the co-culture, washed two times with FACS Stain Buffer (BD), and stained with the indicated antibody mastermix for 20 min at 4 °C. Then, the cells were harvested and fixed with Fix/Perm (eBioscience) and finally analyzed for Celltrace dilution with flow cytometry. Samples were acquired on a BD LSRFortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and data were analyzed using FlowJo v10 (Tree Star Inc., San Carlos, CA, USA). Calculations of percent proliferated T cells were essentially performed according to [30 (link)].

Lapazio L., Braun M, & Grandien K. (2021). H2-M and H2-O as Targeting Vehicles for the MHC Class II Processing Compartment Promote Antigen-Specific CD4+ T Cell Activation. Vaccines, 9(10), 1053.

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Facs stain buffer

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14 protocols using facs stain buffer

Quantifying DR5 Expression in Tumor Cells

Quantification of DR5 Cell Surface Expression

DR5 Antibody Binding Affinity Assay

Maternal Phagocytic Function in Pregnancy

Tumor Cell Isolation and Characterization

Flow Cytometry Gastric DC Analysis

Isolation of CD31+ endothelial cells

Analyzing Kupffer Cell Polarization

Isolation of CD31+ endothelial cells

Flow Cytometric Analysis of Proliferated T Cells

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