Live dead cell dye

Manufactured by Thermo Fisher Scientific

Sourced in Germany

The LIVE/DEAD cell dye is a fluorescent stain used to distinguish live and dead cells in a sample. It binds to nucleic acids, providing a visual indication of cell viability.

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Lab products found in correlation

Anti ifn γ alexa 700, by BD (3 mentions) Anti cd49d, by BD (3 mentions) Anti tnfα pecy7, by BD (3 mentions) Anti cd107a fitc, by BD (3 mentions) Anti il 2 apc, by BD (3 mentions) Brefeldin a, by BD (3 mentions) Anti cd28, by BD (3 mentions) Monensin, by BD (3 mentions) Anti cd3 alexa 780, by Thermo Fisher Scientific (2 mentions) Anti granzyme b v450, by BD (2 mentions) Anti cd8 pe, by BD (2 mentions) Anti cd8 v500, by BD (1 mentions) Anti cd3 pacblue, by BD (1 mentions) Anti cd4 alexa 780, by BD (1 mentions) Human γ globulin, by Merck Group (1 mentions) Lsr fortessa facs analyzer, by BD (1 mentions) Fix perm, by Thermo Fisher Scientific (1 mentions) Anti cd3 cd28 beads, by Thermo Fisher Scientific (1 mentions) Cd3 clone 17a2, by BioLegend (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)

8 protocols using live dead cell dye

Polyfunctional T-cell Profiling by Flow Cytometry

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ICS flow cytometry was done as previously described⁸⁵. In brief, 10⁶ PBMCs were pulsed with autologous peptide at 10 μM in the presence of co-stimulatory antibodies (anti-CD28 and anti-CD49D), anti-CD107a-FITC, monensin, and brefeldin A (all from BD Biosciences) for 6 hrs. The cells were then surface stained with LIVE/DEAD cell dye (Invitrogen), anti-CD3-Alexa 780 (eBioscience), and anti-CD8-PE (BD Biosciences). The cells were permeabilized and labeled with anti-IFN-γ-Alexa 700, anti-IL-2-APC, anti-TNFα-PECy7, and anti-Granzyme B-V450 (all from BD Biosciences). CD3 events greater than 100,000 were acquired on an LSR II (BD Immunocytometry Systems), and data were analyzed using FlowJo (version 9.6.4; TreeStar). Polyfunctionality analysis was performed using Boolean gating and SPICE & PESTLE (version 5.1; NIAID)⁸⁶.

Carlson J.M., Du V.Y., Pfeifer N., Bansal A., Tan V.Y., Power K., Brumme C.J., Kreimer A., DeZiel C.E., Fusi N., Schaefer M., Brockman M.A., Gilmour J., Price M.A., Kilembe W., Haubrich R., John M., Mallal S., Shapiro R., Frater J., Harrigan P.R., Ndung’u T., Allen S., Heckerman D., Sidney J., Allen T.M., Goulder P.J., Brumme Z.L., Hunter E, & Goepfert P.A. (2016). Impact of Pre-adapted HIV Transmission. Nature medicine, 22(6), 606-613.

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Polyfunctional T-cell Immune Response Analysis

Cited 1 time

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Flow cytometry based ICS assay was done as previously described [35 ,49 (link),50 (link)]. In brief, 10⁶ PBMCs were pulsed with peptide at 10uM in the presence of co-stimulatory antibodies (anti-CD28 and anti-CD49D) and anti-CD107a-FITC (all from BD Biosciences) for 2 hrs at 37°C. Monensin and brefeldin A (both from BD Biosciences) were then added and the cultures incubated for an additional 12 hrs. Next day, the cells were labeled with LIVE/DEAD cell dye (Invitrogen) and surface stained with anti-CD3-Pac Blue, anti-CD8-V500, and anti-CD4-Alexa 780 (both from BD Biosciences). The cells were permeabilized and labeled with anti-IFN-γ-Alexa 700, anti-IL-2-APC, anti-TNFα-PECy7, and anti-Granzyme A-PE (all from BD Biosciences). CD3 events greater than 100,000 were acquired on an LSR II (BD Immunocytometry Systems), and data were analyzed using FlowJo (version 9.6.4; TreeStar). Polyfunctionality analysis was performed using Boolean gating and SPICE and Pestle software (version 5.1; NIAID).

Erdmann N., Du V.Y., Carlson J., Schaefer M., Jureka A., Sterrett S., Yue L., Dilernia D., Lakhi S., Tang J., Sidney J., Gilmour J., Allen S., Hunter E., Heath S., Bansal A, & Goepfert P.A. (2015). HLA Class-II Associated HIV Polymorphisms Predict Escape from CD4+ T Cell Responses. PLoS Pathogens, 11(8), e1005111.

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Multiparametric NK cell profiling

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Fresh PBMC or decidual cells were washed and resuspended in 100 ml FACS buffer (PBS, 1% FCS) and incubated with 5 mg human γ-globulins (Sigma-Aldrich) for 15 min to block nonspecific binding. Staining with directly conjugated Abs to surface markers was performed for 25 min at 4°C. Abs were CD56 (clone HCD56, BV421, BioLegend), CD3 (UCHT1, allophycocyanin-Cy7, BioLegend) and CD9-PerCP-Cy5.5 (M-L13, PerCP, BD Pharmingen), KIR2DL1 (143211, FITC, R&D Systems), NKG2A (Z199, allophycocyanin, Beckman Coulter), KIR2DL3/L2/S2 (GL183, PE-Cy7, Beckman Coulter), CD122 (Tu27, APC, BD Biosciences), and LILRB1 (HP-F1, PE, Beckman Coulter). For some donors KIR2DL3 alone was stained using clone 180701 (R&D Systems). KIR3DL1 was stained with DX9 conjugated to biotin (BioLegend). Cells were washed with FACS buffer twice followed by staining the near-IR fixable Live/Dead cell dye (Invitrogen) and biotinylated Ab was detected with streptavidin Qdot-605. After washing in FACS buffer, cells were and fixed in 2% paraformaldehyde. For intracellular Ags, cells were treated with FIX & PERM (Life Technologies) and stained for Ki67 (B56, PerCP-cy5.5, BD Biosciences) or c-Myc (9E10, Alexa647, AbD Serotec). Samples were run on an LSRFortessa FACS analyzer (BD Biosciences) and data were analyzed using FlowJo (Tree Star).

Sharkey A.M., Xiong S., Kennedy P.R., Gardner L., Farrell L.E., Chazara O., Ivarsson M.A., Hiby S.E., Colucci F, & Moffett A. (2015). Tissue-Specific Education of Decidual NK Cells. The Journal of Immunology Author Choice, 195(7), 3026-3032.

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Activation of IL-17 in Mouse Lymphoma EL4 Cells

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Example 65

Determination of the Activation Rate on Mouse Lymphoma EL4

Mouse lymphoma EL4 cells transfected with RORγt plasmid were cultured at 37° C. under an atmosphere with 5% CO₂, with a test compound added simultaneously. After 24 hours, the generation efficiency of IL-17A was analyzed. Before collection of cells, PMA at 50 ng/mL and ionomycin at 500 ng/mL were added for stimulation for 4 hours. The proportion of IL-17 was detected by intracellular staining and flow cytometry. Meanwhile, Live/Dead Cell Dye (Invitrogen) staining was used to analyze the cell survival rate and to judge whether the drug had toxicity to cells. The activation rate of IL-17 generated by EL4 cells for the compound was measured at a concentration of 2 μM. The results showed that the compounds of the present invention had good abilities to increase IL-17 generation (see Table 2).

TABLE 2

Determination of the activation of IL-17 generated by EL4 cells

Example No.₊act %

2**

6**

9A*

11*

13*

16***

17***

19**

20***

21**

24*

26*

32**

33**

34***

35***

36***

37**

40***

41***

47*

49*

53**

55**

58**

60**

62**

*** means ₊act %@ 2 μM > 50%;

** means ₊act %@ 2 μM between 20%-50%;

* means ₊act %@ 2 μM < 20%.

US10844017B2. Biaryl compound, preparation method and use thereof (2020-11-24). FUDAN UNIVERSITY [CN]. Inventors: Yonghui Wang [CN], Yafei Huang [CN], Ruomeng Qiu [CN], Ting Tang [CN].

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Quantifying Th17 Cell Differentiation Potency

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Example 66

Determination of the Half-Maximal Effect Concentration of Mouse Th17 Cell Differentiation

Experimental methods: Mouse spleen CD4⁺T cells were separated and differentiated into Th17 cells. CD4⁺T cells were cultured in the environment containing anti-CD3 (0.25 μg/mL), anti-CD28 (1 μg/mL), anti-IL4 (2 μg/mL), anti-IFN-γ (2 μg/mL), TGF-β (5 ng/mL), and IL6 (20 ng/mL), with a test compound added at the same time. After 96 hours, the differentiation efficiency of Th17 was analyzed. Before collection of cells, PMA at 50 ng/mL and ionomycin at 500 ng/mL were added for stimulation for 4 hours, and the proportion of IL-17 was detected by intracellular staining and flow cytometry. Meanwhile, we used Live/Dead Cell Dye (Invitrogen) staining method to analyze the cell survival rate and to judge whether the drug had toxicity to cells, and determined the half-maximal effect concentration EC₅₀of the compounds. The results showed that the compounds of the present invention had good abilities to induce Th17 differentiation and increase IL-17 production (see Table 3).

TABLE 3

EC₅₀results of mouse Th17 cell differentiation experiments

Example

No.EC₅₀

16***

17***

19**

20*

21***

32***

36*

41*

43**

*** means EC₅₀value < 50 nM;

** means EC₅₀value between 50 nM to 150 nM;

* means EC₅₀value > 150 nM.

US10844017B2. Biaryl compound, preparation method and use thereof (2020-11-24). FUDAN UNIVERSITY [CN]. Inventors: Yonghui Wang [CN], Yafei Huang [CN], Ruomeng Qiu [CN], Ting Tang [CN].

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Polyfunctional T-cell Profiling by Flow Cytometry

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Treg Suppression of CD4+ T Cell Proliferation

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10-week old female BALB/c, NZM, and iNZM mice were treated with/without UVB for 5 consecutive days. The dLNs were processed into a single cell suspension, as described in flow cytometry. CD4⁺CD25⁺ T_Reg cells and CD4⁺ T cells were isolated via CD4+ CD25+ Regulatory T Cell Isolation Kit and CD4+ T Cell Isolation Kit, respectively (Miltenyi Biotec, Bergish Gladbach, Germany). T_Reg cells were labeled with CFSE (ThermoFisher, Eugene, Oregon) and CD4⁺ cells were labeled with cell proliferation dye 670 (ThermoFisher). Following labeling, the cells were co-incubated at a ratio of 0:1,1:1,1:2,1:4 (T_Reg: T_Effector) with/without anti-CD3/CD28 beads (ThermoFisher) for 72 hrs in a 96-well plate. Cells were then stained, as described in the flow cytometry section, for CD3 clone: 17A2 (BioLegend) for 45 minutes, followed by staining with live/dead cell dye (ThermoFisher) for 30 minutes. After staining, cells were resuspended in PBS and data collected on a BD LSR II flow cytometer and analyzed using FlowJo. For analysis, samples were gated on CFSE negative cells to exclude T_Regs, followed by live cell gating, then CD3+ cells and lastly proliferation dye 670 to examine percent proliferation of CD4⁺ T cells (Supplementary figure 1). Percentage proliferation was calculated using the formula: (100 × 1:1,1:2, or 1:4, samples) /0:1 sample.

Wolf S.J., Estadt S.N., Theros J., Moore T., Ellis J., Liu J., Reed T.J., Jacob C.O., Gudjonsson J.E, & Kahlenberg J.M. (2019). Ultraviolet light induces increased T cell activation in lupus-prone mice via type I IFN-dependent inhibition of T regulatory cells. Journal of autoimmunity, 103, 102291.

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Live/Dead Cell Staining Assay

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All cell culture steps refer to Materials and methods 2.18, and only the seeding density was modified to 1 × 10⁵ cells/well. Briefly, the cell culture plate obtained above were washed with PBS three times, and then the live/dead cell dye (R37601, Thermo Fisher Scientific, Germany) was added to the culture plate and incubated in a 37 °C humidified environment with 5% CO₂ for 30 min. After staining, the cell culture plates were washed with PBS three times, and then the stained cells were observed by a fluorescence microscope, where the living cells showed green while the dead cells showed red. The statistical analysis was performed through ImageJ software.

Liu X., Sun Y., Chen B., Li Y., Zhu P., Wang P., Yan S., Li Y., Yang F, & Gu N. (2021). Novel magnetic silk fibroin scaffolds with delayed degradation for potential long-distance vascular repair. Bioactive Materials, 7, 126-143.

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