In-vitro cytotoxicity was assessed using K562 tumor targets at a 10:1 E:T ratio as previously described (43 (link)). Intracellular granzyme B and CD107a expression was measured by flow cytometry as previously described (43 (link)). Briefly, PBMCs were mixed with RPMI1640 medium or target cells at a ratio of 10:1 in the absence of exogenous cytokines in medium. For CD107a expression, anti-CD107a-FITC (Miltenyi Biotech) was added to each well and incubated for 1 hour at 37°C. After 1 hour, brefeldin A (Golgi Plug; BD Biosciences) was added to each well, and the cells were incubated for additional 3 hours. Cells were then washed, fixed, permeabilized using Cytofix/Cytoperm reagent kit (BD Biosciences), and resuspended in staining buffer containing anti-CD56-PE-Vio770 (Miltenyi Biotech). For Granzyme B levels, cells were incubated for 4 hours, washed, fixed, permeabilized using a Cytofix/Cytoperm reagent kit (BD Biosciences), and resuspended in staining buffer containing anti-CD56-PE-Vio770 (Miltenyi Biotech) and granzyme B-FITC (Miltenyi Biotech).
Anti cd56 pe vio770
Manufactured by Miltenyi Biotec
Anti-CD56-PE-Vio770 is a fluorochrome-conjugated antibody that binds to the CD56 antigen. CD56 is a cell surface glycoprotein expressed on natural killer cells, a subset of T cells, and some neuronal cells. The PE-Vio770 fluorochrome allows for the detection and analysis of CD56-positive cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Golgiplug, by BD (1 mentions)
Cytofix cytoperm reagent kit, by BD (1 mentions)
Granzyme b fitc, by Miltenyi Biotec (1 mentions)
Anti cd107a fitc, by Miltenyi Biotec (1 mentions)
Anti cd3 alexafluor700, by BioLegend (1 mentions)
Zombie uv viability dye, by BioLegend (1 mentions)
Anti cd4 alexa fluor 488, by BioLegend (1 mentions)
2 protocols using anti cd56 pe vio770
1
Cytotoxicity and Cytotoxic Effector Function Assessment
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In-vitro cytotoxicity was assessed using K562 tumor targets at a 10:1 E:T ratio as previously described (43 (link)). Intracellular granzyme B and CD107a expression was measured by flow cytometry as previously described (43 (link)). Briefly, PBMCs were mixed with RPMI1640 medium or target cells at a ratio of 10:1 in the absence of exogenous cytokines in medium. For CD107a expression, anti-CD107a-FITC (Miltenyi Biotech) was added to each well and incubated for 1 hour at 37°C. After 1 hour, brefeldin A (Golgi Plug; BD Biosciences) was added to each well, and the cells were incubated for additional 3 hours. Cells were then washed, fixed, permeabilized using Cytofix/Cytoperm reagent kit (BD Biosciences), and resuspended in staining buffer containing anti-CD56-PE-Vio770 (Miltenyi Biotech). For Granzyme B levels, cells were incubated for 4 hours, washed, fixed, permeabilized using a Cytofix/Cytoperm reagent kit (BD Biosciences), and resuspended in staining buffer containing anti-CD56-PE-Vio770 (Miltenyi Biotech) and granzyme B-FITC (Miltenyi Biotech).
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In-vitro cytotoxicity was assessed using K562 tumor targets at a 10:1 E:T ratio as previously described (43 (link)). Intracellular granzyme B and CD107a expression was measured by flow cytometry as previously described (43 (link)). Briefly, PBMCs were mixed with RPMI1640 medium or target cells at a ratio of 10:1 in the absence of exogenous cytokines in medium. For CD107a expression, anti-CD107a-FITC (Miltenyi Biotech) was added to each well and incubated for 1 hour at 37°C. After 1 hour, brefeldin A (Golgi Plug; BD Biosciences) was added to each well, and the cells were incubated for additional 3 hours. Cells were then washed, fixed, permeabilized using Cytofix/Cytoperm reagent kit (BD Biosciences), and resuspended in staining buffer containing anti-CD56-PE-Vio770 (Miltenyi Biotech). For Granzyme B levels, cells were incubated for 4 hours, washed, fixed, permeabilized using a Cytofix/Cytoperm reagent kit (BD Biosciences), and resuspended in staining buffer containing anti-CD56-PE-Vio770 (Miltenyi Biotech) and granzyme B-FITC (Miltenyi Biotech).
2
Donor Leukocyte Profiling in Transplant
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Flow cytometry was used to determine the donor leukocyte profile in both the graft and perfusate compartments. Isolated cells were washed and stained with Zombie UV viability dye (Biolegend, San Diego, CA) in phosphate-buffered saline and then stained with the following markers: anti-CD3 Alexa Fluor 700 (Biolegend), anti-CD4 Alexa Fluor 488 (Biolegend), anti-CD8 Brilliant Violet 785 (Biolegend), anti-CD14 Brilliant Violet 650 (Biolegend), anti-CD16 Brilliant Violet 570 (Biolegend), anti-CD19 Brilliant Violet 510 (Biolegend), anti-CD45 Alexa Fluor 532 (Invitrogen, Carlsbad, CA), anti-CD56 PE-Vio770 (Miltenyl Biotec, Bergisch Gladbach, Germany), and anti-CD66b PE-dazzle (Biolegend) (Supplemental Table 1 Supplemental Figure 1
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