Dissected spleens were ground on a pre-wet 70 μm filter with a 2 ml syringe plunger. The filters were washed with 10 ml PBS and cell suspension centrifuged at 300 × g for 5 min at 4 °C. The pellet was resuspended in 3 ml Pharm Lyse red blood cell lysis buffer (BD) and incubated for 5 min at room temperature. Ten ml Flow-cytometry buffer (PBS + 1% BSA + 0.05% NaN3) was used to stop the reaction, followed by another centrifugation step. Cell suspension was filtered through a 30 μm filter and cells were counted using Tali chips (Thermo Fisher Scientific). Fc receptors of cells were blocked using 1:50 anti-CD16/32 (clone 93, BioLegend). Around 1 × 106 cells were stained with BV605 anti-CD3 (clone 17A2, BioLegend), BV480 anti-CD4 (clone RM4-5, BD), PerCP-Cy5.5 anti-CD8 (clone 53–6.7, BioLegend), BUV395 anti-CD44 (clone IM7, BD) and PE anti-CD62L (clone MEL-14, BioLegend). The cells were incubated for 60 min on ice and washed twice with staining buffer. A 1:10 DAPI solution was used to distinguish live and dead cells. Flow-cytometry data were acquired on a BD Fortessa analyser and analysed using FCS Express (V6, De Novo Software). Please see gating scheme in Supplementary Fig. 2 .
Pe anti cd62l clone mel 14
Manufactured by BioLegend
PE-anti-CD62L (clone MEL-14) is a flow cytometry reagent used for the detection and quantification of CD62L (L-selectin) expression on the surface of cells. CD62L is a cell adhesion molecule involved in the homing of lymphocytes to lymphoid tissues. This reagent is conjugated with the fluorescent label phycoerythrin (PE) for sensitive detection by flow cytometry.
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Lab products found in correlation
Moflo facs sorter, by Agilent Technologies (2 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (2 mentions)
Anti cd44 fitc clone im7, by BioLegend (2 mentions)
Il 23 il 2, by Thermo Fisher Scientific (2 mentions)
Cd25 mab, by Thermo Fisher Scientific (2 mentions)
Il 23, by Thermo Fisher Scientific (2 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (2 mentions)
Fcs express, by De Novo Software (1 mentions)
Anti cd16 32 clone 93, by BioLegend (1 mentions)
Fortessa analyser, by BD (1 mentions)
3 protocols using pe anti cd62l clone mel 14
1
Splenic Cell Isolation and Characterization
2
Isolation and Characterization of Memory Th17 Cells
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Draining lymph nodes from DED mice were harvested and pooled, and CD4+ T cells were isolated via negative selection with a CD4+ T cell isolation kit (Miltenyi Biotec Inc.). The isolated cells were >98% CD4+ as confirmed by flow cytometry, and they were stained with FITC-anti-CD44 (clone IM7) and PE-anti-CD62L (clone MEL-14, BioLegend) antibodies, and sorted for CD44loCD62L- effector subpopulation using a MoFlo® FACS sorter (Dako Cytomation). The sorted CD44loCD62L-CD4+ cells were treated with IL-23 (10 ng/ml, eBioscience), IL-2 (50 IU/ml, Pepro Tech), IL-23+IL-2, monoclonal CD25 blocking Ab (CD25 mAb, 10 μg/ml, clone PC61.5, eBioscience), or IL-23+CD25 mAb for 48 hours. Memory Th17 cells, as well as expression levels of IL-7R, IL-15R, and Annexin V were then examined by flow cytometry.
3
Isolation and Characterization of Memory Th17 Cells
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