For phenotype analysis, cells were stained with 7AAD (Beckman) to identify viable cells and antibodies against the surface markers CD25-FITC, CD45RO-FITC, CD69-PE, CD62L-PE, CD19-PE, CD3-PE, CD19-ECD, CD56-PECy7, CD56-APC, CD3-APC, CD45-APCAlexaFluor750, CD45RA-APCAlexaFluor750, CD16-PacificBlue, CD57-PacificBlue, CD45-KromeOrange, CD16-KromeOrange (Beckman), CD158b-FITC, CD158a-PE, CD107a-HV500 (BD Biosciences), CD158e-Vioblue (Miltenyi). 1×105-3×105 cells were incubated for 20-30 min at 4 °C with different antibodies in PBS containing 2.5% FBS. Cells were then washed and suspended in 200-250 µL of the same media. Staining was analyzed on a Gallios flow cytometer (Beckman) using the Kaluza software. Alive lymphocytes were gated using FSC/SSC and 7AAD staining. B lymphocytes (CD19+), T lymphocytes (CD3+CD56-) and NK cells (CD56+CD3-) were distinguished using, respectively, CD19, CD3 and CD56 antibodies.
Cd107a hv500
Manufactured by BD
CD107a-HV500 is a fluorochrome-conjugated monoclonal antibody that can be used for the flow cytometric detection and analysis of CD107a (also known as LAMP-1) expression on cell surfaces. CD107a is a marker of degranulation and is commonly used to assess the cytotoxic activity of natural killer cells and cytotoxic T cells.
Automatically generated - may contain errors
Lab products found in correlation
Gallios flow cytometer, by Beckman Coulter (3 mentions)
Cd3 apc, by BD (3 mentions)
Cd56 pe cy7, by BD (3 mentions)
Cd45ra apcalexafluor750, by BD (3 mentions)
7 aad, by Beckman Coulter (2 mentions)
Cd19 pe, by BD (2 mentions)
Monensin, by BD (2 mentions)
Count and viability kit, by Merck Group (2 mentions)
Muse cytometer, by Merck Group (2 mentions)
Cd16 kromeorange, by BD (2 mentions)
Cd45ro fitc, by BD (2 mentions)
Cd158b fitc, by BD (1 mentions)
Cd57 pacificblue, by Beckman Coulter (1 mentions)
Cd45 krome orange, by Beckman Coulter (1 mentions)
Cd69 pe, by BD (1 mentions)
Anti cd107a antibody, by BD (1 mentions)
Cd19 ecd, by BD (1 mentions)
Facs gallios flow cytometer, by Beckman Coulter (1 mentions)
Anti cd45ro fitc, by BD (1 mentions)
4 protocols using cd107a hv500
1
Multiparametric Flow Cytometry Analysis
2
NK Cell Degranulation Assay Protocol
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This was done as previously described (15 (link)). Briefly, 50 × 103 target cells per well were placed in RPMI, 10% FBS, IL-2 100 U/mL with monensin (BD Biosciences) in a 96-well V-bottom plate. NK and target cells were incubated overnight at 37°C in 5% CO2, and living cells were counted using a Muse cytometer (Millipore) with a count and viability kit (Millipore). As a control, NK cells were incubated without target cells. CD107a+ NK cells were analyzed on a Gallios flow cytometer (Beckman Coulter) using 7AAD, CD45RO-FITC, CD19-PE, CD56-PECy7, CD3-APC, CD45RA-APCAlexaFluor750, CD16-KromeOrange, and CD107a-HV500 (BD Biosciences). The results were analyzed using Kaluza software.
3
NK Cell Cytotoxicity Assay with K562 Targets
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After PBMC purification and NK cell quantification, 3 million cells were incubated at 37°C for 4h or overnight with K562 target cells at an Effector (NK cell): Target ratio of 1:10 in a final volume of 500μl (RPMI Glutamax with 10% FBS and 10u/ml IL2). The medium also contained 1.5μl anti-CD107a antibody (BD Biosciences, Franklin Lakes, NJ) and 1μl monensin to prevent CD107a degradation (BD Golgi-Stop BD Biosciences). Then, cells were resuspended in 50μl of an antibody cocktail containing 7AAD, the anti-CD45RO-FITC, -CD69-PE, -CD19-ECD, -CD56-PECy7, -CD3-APC, -CD45RA-APCAlexaFluor750, -CD107a-HV500 and -CD16-KO antibodies (BD Biosciences, Beckman). Samples were analyzed on a Beckman Coulter FACS Gallios flow cytometer using the Kaluza software. Events were initially gated on forward and side scatter (SSC) to identify lymphocytes. A bivariate plot of CD56 versus CD3 was used to acquire at least 10,000 NK cells.
4
NK Cell Cytotoxicity Assay
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Briefly, 50×103 target cells per well were placed in RPMI, 10% FBS, IL-2 100 U/mL with monensin (BD Biosciences) in a 96-well V-bottom plate. NK and target cells were incubated overnight at 37°C in 5% CO2 and living cells were counted using a Muse cytometer (Millipore) with the count and viability kit (Millipore). As a control, NK cells were incubated without targets. CD107a+ NK cells were analyzed on a Gallios flow cytometer (Beckman Coulter) using 7AAD, CD45RO-FITC, CD19-PE, CD56-PECy7, CD3-APC, CD45RA-APCAlexaFluor750, CD16-KromeOrange and CD107a-HV500 (BD Biosciences). Results were analyzed using Kaluza software.
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