We repurposed an intravascular cell labeling technique, as described previously39 (link), to assess availability of antibody to vascular and parenchymal T cells. Briefly, mice were injected intravenously with biotin-conjugated anti-CD8α. Three minutes after the injection, mice were euthanized and tissues were harvested as described10 (link),36 (link). In brief, FRT tissue was removed, digested in Collagenase IV (Sigma) with DNAse for 1 hour, then dissociated via gentleMACS Dissociator (Miltenyi Biotec), and lymphocytes were purified on a 44/67% Percoll (GE Healthcare) gradient. Isolated cells were stained with Steptavidin-BV650 (Biolegend) to label biotin-CD8α bound cells, and antibodies to CD44 (clone IM7, BioLegend), CD45.1 (clone A20, Biolegend), Thy1.1 (Clone OX7, Biolegend), and CD8β (clone Ly-3, Biolegend). All cells were stained at antibody dilutions of 1:100 except Thy1.1, which was 1:1000. Cell viability was determined with Ghost Dye 780 (Tonbo Biosciences). The stained samples were acquired with LSRII or LSR Fortessa flow cytometers (BD Biosciences) and analyzed with FlowJo software (Treestar).
Steptavidin bv650
Manufactured by BioLegend
Streptavidin-BV650 is a fluorescent protein conjugate used in flow cytometry and other immunoassays. It binds to biotin with high affinity, allowing it to be utilized as a detection reagent for biotinylated antibodies or other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Cd44 clone im7, by BioLegend (2 mentions)
Cd45.1 clone a20, by BioLegend (2 mentions)
Cd8β clone ly 3, by BioLegend (2 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (2 mentions)
Collagenase 4, by Merck Group (2 mentions)
Ghost dye 780, by Cytek Biosciences (2 mentions)
Thy1.1 clone ox 7, by BioLegend (2 mentions)
Lsrii or lsr fortessa flow cytometers, by BD (2 mentions)
2 protocols using steptavidin bv650
1
Intravascular T Cell Labeling Protocol
2
Intravascular T Cell Labeling Protocol
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We repurposed an intravascular cell labeling technique, as described previously39 (link), to assess availability of antibody to vascular and parenchymal T cells. Briefly, mice were injected intravenously with biotin-conjugated anti-CD8α. Three minutes after the injection, mice were euthanized and tissues were harvested as described10 (link),36 (link). In brief, FRT tissue was removed, digested in Collagenase IV (Sigma) with DNAse for 1 hour, then dissociated via gentleMACS Dissociator (Miltenyi Biotec), and lymphocytes were purified on a 44/67% Percoll (GE Healthcare) gradient. Isolated cells were stained with Steptavidin-BV650 (Biolegend) to label biotin-CD8α bound cells, and antibodies to CD44 (clone IM7, BioLegend), CD45.1 (clone A20, Biolegend), Thy1.1 (Clone OX7, Biolegend), and CD8β (clone Ly-3, Biolegend). All cells were stained at antibody dilutions of 1:100 except Thy1.1, which was 1:1000. Cell viability was determined with Ghost Dye 780 (Tonbo Biosciences). The stained samples were acquired with LSRII or LSR Fortessa flow cytometers (BD Biosciences) and analyzed with FlowJo software (Treestar).
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