Live dead fixable violet dead stain kit, by Thermo Fisher Scientific - Product details

1

HUVEC Inflammatory Response Characterization

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Human umbilical vascular endothelial cells (HUVECs) were purchased from American Tissue Type Collection. Endothelial Growth Medium (EGM) 2 was obtained from Lonza. AlexaFluor 647-conjugated anti-ICAM1, phycoerythryin-conjugated anti-VCAM1, anti-CXCL10, phycoerythryin/Cy7-conjugated anti-CD14 antibodies and the protein transport inhibitor monensin were purchased from BioLegend. Phycoerythryin-Cy5-conjugated anti-E-selectin and APC-conjugated anti-CD19 antibodies were obtained from BD Biosciences. Qdot 605-conjugated anti-CD3 and Live/Dead fixable violet dead stain kit were from Life Technologies. Recombinant human TNFα and IFNγ, and CXCL10 Quantikine ELISA kit were purchased from R & D Systems.

Xie Z., Chan E., Yin Y., Ghosh C.C., Wisch L., Nelson C., Young M., Parikh S.M, & Druey K.M. (2014). Inflammatory Markers of the Systemic Capillary Leak Syndrome (Clarkson Disease). Journal of clinical & cellular immunology, 5, 1000213-.

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2

Detecting Apoptosis in IL-4 and Anti-CD40 Cultured Cells

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To validate some of the differentially expressed genes we fixed, permeabilized, and stained cells as previously described (7 (link)). The antibodies used were as follows; anti-IL4R APC (R&D), anti-CD27 FITC (Biolegend), anti-CD38 PE-CY7 (Biolegend), anti-CD20 FITC (Biolegend), anti-IRF4 alexa 647 (Invitrogen), anti-IRF8 APC (Biolegend), anti-BLIMP1 APC (R&D), and anti-active Caspase 3 alexa 647 (BD Biosciences). To determine the rates of apoptosis the IL-4 and anti-CD40 cultured cells were harvested and the dead cells removed using the Easysep dead cell removal kit (Stemcell). The cells were then recultured for 24 h with IL-4 and anti-CD40, followed by staining for Annexin V (eBioscience) and live/dead fixable violet dead stain kit (Life Technologies). Data was collected on a BD FACSCanto (BD Biosciences) and events were analyzed using FlowJo software version 10.4.2 (Tree Star).

Ramadani F., Bowen H., Gould H.J, & Fear D.J. (2019). Transcriptional Analysis of the Human IgE-Expressing Plasma Cell Differentiation Pathway. Frontiers in Immunology, 10, 402.

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3

HUVEC Inflammatory Response Characterization

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Human umbilical vascular endothelial cells (HUVECs) were purchased from American Tissue Type Collection. Endothelial Growth Medium (EGM) 2 was obtained from Lonza. AlexaFluor 647-conjugated anti-ICAM1, phycoerythryin-conjugated anti-VCAM1, anti-CXCL10, phycoerythryin/Cy7-conjugated anti-CD14 antibodies and the protein transport inhibitor monensin were purchased from BioLegend. Phycoerythryin-Cy5-conjugated anti-E-selectin and APC-conjugated anti-CD19 antibodies were obtained from BD Biosciences. Qdot 605-conjugated anti-CD3 and Live/Dead fixable violet dead stain kit were from Life Technologies. Recombinant human TNFα and IFNγ, and CXCL10 Quantikine ELISA kit were purchased from R & D Systems.

Xie Z., Chan E., Yin Y., Ghosh C.C., Wisch L., Nelson C., Young M., Parikh S.M, & Druey K.M. (2014). Inflammatory Markers of the Systemic Capillary Leak Syndrome (Clarkson Disease). Journal of clinical & cellular immunology, 5, 1000213-.

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4

Myeloid Cell Phenotyping with DCFDA

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The viably cryopreserved PBMC were thawed and stained with LIVE/DEAD fixable violet Dead Stain Kit (Thermo Fisher Scientific) for 20 minutes followed by staining with a myeloid panel of antibodies comprising CD14-APC-Cy7 (564123), CD33-PE-Cy7 (333952), CD16-BV605 (563172), HLA-DR-BV786 (564041), all from BD Biosciences, for 30 minutes at 4°C. Cells were then washed and stained with H2-DCFDA (D399, Molecular Probes) in serum-free Iscoves’ Modified Dulbecco's Medium (IMDM; 36531, Thermo Fisher Scientific) for 30 minutes at 37°C, washed and acquired on a five laser BD LSR Fortessa and analyzed with FlowJo.

Grauers Wiktorin H., Aydin E., Kiffin R., Vilhav C., Bourghardt Fagman J., Kaya M., Paul S., Westman B., Bratlie S.O., Naredi P., Hellstrand K, & Martner A. (2024). Impact of Surgery-Induced Myeloid-derived Suppressor Cells and the NOX2/ROS Axis on Postoperative Survival in Human Pancreatic Cancer. Cancer Research Communications, 4(4), 1135-1149.

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5

Flow Cytometry Analysis of Irradiated Tumor Cells

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At 96 h post-irradiation, tumor cells were examined by flow cytometry, as previously described [13 (link)]. Cells were examined on FACSCalibur or FACSVerse cytometers, using the monoclonal antibodies targeting HLA-ABC-FITC, HLA-ABC-PE-Cy7, ICAM-1 (CD54)-PE, CEA (CD66)-FITC, MUC-1 (CD227)-FITC, CD24-PerCP-Cy5.5, CD44-FITC, and the appropriate isotype-matched controls (BD Biosciences, San Jose, CA). The monoclonal antibodies targeting CD70-FITC, CD275 (ICOSL)-PE, CD137L (4-1BBL)-PE, CD252 (OX40L)-PE, PD-L1 (CD274)-PE, CTLA-4 (CD152)-PE, and CD227 (MUC-1)-PE were obtained from BioLegend (San Diego, CA). Antibodies targeting CD133-APC (Miltenyi Biotec, San Diego, CA) and calreticulin-PE (R&D Systems, Minneapolis, MN) were also used. Isotype control staining was < 5% for all samples analyzed. Viability was examined using LIVE/DEAD Fixable Violet Dead Stain Kit (Thermo Fisher Scientific, Rockville, MD). Cell surface expression was evaluated on live cells gated by FSC/SSC and LIVE/DEAD staining.

Gameiro S.R., Malamas A.S., Bernstein M.B., Tsang K.Y., Vassantachart A., Sahoo N., Tailor R., Pidikiti R., Guha C.P., Hahn S.M., Krishnan S, & Hodge J.W. (2016). Tumor Cells Surviving Exposure to Proton or Photon Radiation Share a Common Immunogenic Modulation Signature, Rendering Them More Sensitive to T Cell-Mediated Killing. International journal of radiation oncology, biology, physics, 95(1), 120-130.

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6

CRISPR Library Sorting Protocol

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For CRISPR library sorting, > 10⁸ cells were taken for antibody staining (10,000 library representation). We set aside 10⁷ cells for the pre-sort control (1,000 coverage). After harvesting the cells and removing leftover medium by washing with PBS, the cells were stained for 5 minutes at room temperature with LIVE/DEAD Fixable Violet Dead Stain Kit (ThermoFisher L34864). Subsequently, the cells were stained with antibodies for 20 minutes on ice. The following antibodies were used: CD45-PE (clone 2D1), CD46-APC (clone TRA-2–10) or CD55-APC (clone JS11). All antibodies were purchased pre-conjugated from BioLegend. Cells were washed with PBS to remove unbound antibodies prior to sorting. Cell acquisition and sorting was performed using a Sony SH800S cell sorter.
Sequential gating was performed as follows: 1) exclusion of debris based on forward and side scatter cell parameters, 2) doublet exclusion, and 3) dead cell exclusion (Figure S1C). The sorting gates were set based on the expression level of the target protein in sgRNA library-transduced cells (top and bottom 15% of expression, Figure S1E). Typically, we achieved >500 library coverage within each sorted population.

Legut M., Daniloski Z., Xue X., McKenzie D., Guo X., Wessels H.H, & Sanjana N.E. (2020). High-Throughput Screens of PAM-Flexible Cas9 Variants for Gene Knockout and Transcriptional Modulation. Cell reports, 30(9), 2859-2868.e5.

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7

CRISPR Library Sorting Protocol

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For CRISPR library sorting, > 10⁸ cells were taken for antibody staining (10,000 library representation). We set aside 10⁷ cells for the pre-sort control (1,000 coverage). After harvesting the cells and removing leftover medium by washing with PBS, the cells were stained for 5 minutes at room temperature with LIVE/DEAD Fixable Violet Dead Stain Kit (ThermoFisher L34864). Subsequently, the cells were stained with antibodies for 20 minutes on ice. The following antibodies were used: CD45-PE (clone 2D1), CD46-APC (clone TRA-2–10) or CD55-APC (clone JS11). All antibodies were purchased pre-conjugated from BioLegend. Cells were washed with PBS to remove unbound antibodies prior to sorting. Cell acquisition and sorting was performed using a Sony SH800S cell sorter.
Sequential gating was performed as follows: 1) exclusion of debris based on forward and side scatter cell parameters, 2) doublet exclusion, and 3) dead cell exclusion (Figure S1C). The sorting gates were set based on the expression level of the target protein in sgRNA library-transduced cells (top and bottom 15% of expression, Figure S1E). Typically, we achieved >500 library coverage within each sorted population.

Legut M., Daniloski Z., Xue X., McKenzie D., Guo X., Wessels H.H, & Sanjana N.E. (2020). High-Throughput Screens of PAM-Flexible Cas9 Variants for Gene Knockout and Transcriptional Modulation. Cell reports, 30(9), 2859-2868.e5.

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Live dead fixable violet dead stain kit

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7 protocols using live dead fixable violet dead stain kit

HUVEC Inflammatory Response Characterization

Detecting Apoptosis in IL-4 and Anti-CD40 Cultured Cells

HUVEC Inflammatory Response Characterization

Myeloid Cell Phenotyping with DCFDA

Flow Cytometry Analysis of Irradiated Tumor Cells

CRISPR Library Sorting Protocol

CRISPR Library Sorting Protocol

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