For intracellular cytokine staining, 2 × 106 splenocytes were stimulated in the presence of protein transport inhibitor, GolgiStopTM GolgiPlugTM (BD Bioscience) with the same peptide pools as the ELISpots. Media alone and phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulations (BD Bioscience) were used as negative and positive controls respectively. To test for degranulation of cells, FITC anti-CD107a (Biolegend) antibody was also added during stimulation. After 6 h, cells were washed and stained with LIVE/DEAD violet. Surface staining was then added containing BV510 anti-CD4, APC-Cy7 anti-CD8, BV711 anti-CD62L, and AF700 anti-CD44 (Biolegend). After 30 min incubation, cells were spun, washed, and fixed using the CytoPerm CytoWash kit following the manufacturer’s protocol (BD Bioscience). Intracellular staining was then prepared using APC anti-IFNγ, BV650 anti-TNFα, PE-Cy7 anti-IL-2, and PE-Cy5 anti-CD3 (Biolegend). All data were collected on a modified LSRII flow cytometer (BD Bioscience) with FACS DIVA 6.0 GUI followed by analysis with FlowJo software 9.0 (BD Bioscience).
Af700 anti cd44
Manufactured by BioLegend
AF700 anti-CD44 is a fluorescently-conjugated antibody that binds to the CD44 cell surface receptor. CD44 is a glycoprotein involved in cell-cell interactions, cell adhesion, and migration. The AF700 fluorescent label allows for flow cytometric analysis of CD44 expression on cells.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin stimulations, by BD (2 mentions)
Golgiplug golgistop, by BD (2 mentions)
Fitc anti cd107a, by BioLegend (2 mentions)
Bv711 anti cd62l, by BioLegend (2 mentions)
Cytoperm cytowash kit, by BD (2 mentions)
Bv650 anti tnfα, by BioLegend (2 mentions)
Pe cy7 anti il 2, by BioLegend (2 mentions)
Anti ifn γ apc, by BioLegend (2 mentions)
Pe cy5 anti cd3, by BioLegend (2 mentions)
Anti cd8 apc cy7, by BioLegend (2 mentions)
Anti cd4 bv510, by BioLegend (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Facsdiva 6, by BD (1 mentions)
2 protocols using af700 anti cd44
1
Intracellular Cytokine Profiling of Splenocytes
2
Intracellular Cytokine Staining of Splenocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
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For intracellular cytokine staining, 2x10 6 splenocytes were stimulated in the presence of protein transport inhibitor, GolgiStop TM GolgiPlug TM (BD Bioscience) with the same peptide pools as the ELISpots.
Media alone and phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulations (BD Bioscience) were used as negative and positive controls respectively. To test for degranulation of cells, FITC anti-CD107a (Biolegend) antibody was also added during stimulation. After 6hrs, cells were washed and stained with LIVE/DEAD violet. Surface staining was then added containing BV510 anti-CD4, APC-Cy7 anti-CD8, BV711 anti-CD62L and AF700 anti-CD44 (Biolegend). After 30 minutes incubation, cells were spun, washed, and fixed using the CytoPerm CytoWash kit following manufacturer's protocol (BD Bioscience).
Intracellular staining was then prepared using APC anti-IFNγ, BV650 anti-TNFα, PE-Cy7 anti-IL2, and PE-Cy5 anti-CD3 (Biolegend). All data was collected on a modified LSRII flow cytometer (BD Bioscience) followed by analysis with FlowJo software (BD Bioscience).
Media alone and phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulations (BD Bioscience) were used as negative and positive controls respectively. To test for degranulation of cells, FITC anti-CD107a (Biolegend) antibody was also added during stimulation. After 6hrs, cells were washed and stained with LIVE/DEAD violet. Surface staining was then added containing BV510 anti-CD4, APC-Cy7 anti-CD8, BV711 anti-CD62L and AF700 anti-CD44 (Biolegend). After 30 minutes incubation, cells were spun, washed, and fixed using the CytoPerm CytoWash kit following manufacturer's protocol (BD Bioscience).
Intracellular staining was then prepared using APC anti-IFNγ, BV650 anti-TNFα, PE-Cy7 anti-IL2, and PE-Cy5 anti-CD3 (Biolegend). All data was collected on a modified LSRII flow cytometer (BD Bioscience) followed by analysis with FlowJo software (BD Bioscience).
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