Tetramer enrichment was performed similarly as previously described40 (link), 46 (link). Single-cell suspensions were prepared from the spleens or thymi harvested from wild-type littermate and Sult2b1-deficient mice. Red blood cells (RBCs) were then lysed with ACK buffer. With equal numbers of total cells from each groups, PE-conjugated HY Uty-WMHHNMDLI:Db tetramers (MBLI) were added to the cell suspension at a 1:10 dilution. Cells were incubated for 1 h at 4 °C co-stained with anti-CD8. After washing, cells were resuspended in PBS, 2 mM EDTA and 0.5% BSA, and anti-PE microbeads (Miltenyi Biotec) were added to the cell suspension. Samples were incubated at 4 °C for 15 min and then washed. Cells were applied to pre-washed MACS LS columns. Following three washes, the bound fraction was eluted and resuspended in FACS buffer (PBS, 2 mM EDTA and 5% FBS). Tetramer-bound fractions were stained with 1:100 dilution of fluorophore-conjugated antibodies to the following markers: F4/80 (BM8), Gr-1 (RB6-8C5), CD11b (M1/70), CD11c (N418), Ter119 (TER-119), CD19 (6D5), CD4 (RM4-5) and Live/Dead Aqua amine (Life Technologies). After staining, the washed samples were collected on a BD LSR II and analyzed using FlowJo (Tree Star).
Live dead aqua amine
Manufactured by Thermo Fisher Scientific
The LIVE/DEAD Aqua Amine is a reagent used to stain cells and detect cell viability. It is designed to identify dead cells in a sample by labeling them with a fluorescent dye.
Automatically generated - may contain errors
Lab products found in correlation
Lsr 2, by BD (2 mentions)
Anti pe microbeads, by Miltenyi Biotec (2 mentions)
Foxp3 fix perm kit, by Thermo Fisher Scientific (2 mentions)
Cd19 bv510, by BioLegend (2 mentions)
Cd3 apc cy7, by BioLegend (1 mentions)
Cd4 percp, by BioLegend (1 mentions)
Brilliant violet 421 cd25, by BioLegend (1 mentions)
Cd8 brilliant violet 510, by BioLegend (1 mentions)
Lsrii four laser flow cytometer, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Pe anti siglec f e50 2440, by BD (1 mentions)
Efluor450 anti cd11b m1 70, by Thermo Fisher Scientific (1 mentions)
Cd14 bv510, by BioLegend (1 mentions)
Ifn γ pe cy7, by BioLegend (1 mentions)
Monensin, by BD (1 mentions)
Brefeldin a, by BD (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
6 protocols using live dead aqua amine
1
Tetramer-based Enrichment of Immune Cells
2
Tetramer-based Enrichment of Immune Cells
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Tetramer enrichment was performed similarly as previously described40 (link), 46 (link). Single-cell suspensions were prepared from the spleens or thymi harvested from wild-type littermate and Sult2b1-deficient mice. Red blood cells (RBCs) were then lysed with ACK buffer. With equal numbers of total cells from each groups, PE-conjugated HY Uty-WMHHNMDLI:Db tetramers (MBLI) were added to the cell suspension at a 1:10 dilution. Cells were incubated for 1 h at 4 °C co-stained with anti-CD8. After washing, cells were resuspended in PBS, 2 mM EDTA and 0.5% BSA, and anti-PE microbeads (Miltenyi Biotec) were added to the cell suspension. Samples were incubated at 4 °C for 15 min and then washed. Cells were applied to pre-washed MACS LS columns. Following three washes, the bound fraction was eluted and resuspended in FACS buffer (PBS, 2 mM EDTA and 5% FBS). Tetramer-bound fractions were stained with 1:100 dilution of fluorophore-conjugated antibodies to the following markers: F4/80 (BM8), Gr-1 (RB6-8C5), CD11b (M1/70), CD11c (N418), Ter119 (TER-119), CD19 (6D5), CD4 (RM4-5) and Live/Dead Aqua amine (Life Technologies). After staining, the washed samples were collected on a BD LSR II and analyzed using FlowJo (Tree Star).
3
Multiparametric Flow Cytometry for T-Cell Analysis
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CBMCs and maternal PBMCs were thawed, aliquoted at 1 × 106 cells, cells were washed, stained, fixed, and permeabilized, as per the manufacturer’s instructions (FoxP3 Fix/Perm kit; eBiosciences) using standard protocols using the following antibodies: APC/Cy7 CD3 (clone OKT3), PerCP CD4 (clone RPA-T4), Brilliant Violet 421 CD25 (clone BC96), Brilliant Violet 650 CD127 (clone A019D5), Brilliant Violet 605 CD45RO (clone UCHL1), FITC CCR7 (clone G043H7), Brilliant Violet 510 CD8 (clone SK1), Brilliant Violet 510 CD14 (clone M5E2), Brilliant Violet 510 CD19 (clone HIB19, BioLegend), FITC Ki67 (clone Ki67, BD Pharmingen), PE FoxP3 (clone PCH101, eBioscience), and LIVE/DEAD aqua amine (Invitrogen). Flow cytometry data were collected on an LSR II four-laser flow cytometer (BD) with FACSDiva software. Color compensation was performed using compensation beads. Fluorescence-minus-one samples were used to define negative and positive populations. Cellular profiles were gated on live, single-cell, dump negative (CD14, CD19, CD8), CD3 + lymphocytes.
4
Identifying Immune Cell Subsets via Flow Cytometry
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We used flow cytometry to identify neutrophils (Ly6G+; CD11b+), monocytes (Ly6G-; CD11b+), and eosinophils (SSChigh; Siglec-F+), in air pouch exudates. Red blood cells were lysed by treatment with pH 7.3 ACK lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM EDTA, pH 8.0) 2 times for 5 min each. Cells were blocked with unconjugated anti—CD16/CD32 antibodies (BioXcell) on ice for 5 min and then stained with APC/Cy7 anti-Ly6G (1A8, BD Biosciences), PE anti-Siglec-F (E50-2440, BD Biosciences) and eFluor450 anti-CD11b (M1/70, eBioscience) antibodies on ice for 30 min. Data were acquired using a FACSCanto II flow cytometer (BD Biosciences). Data were analyzed with FlowJo 9.5.3. software (TreeStar). Dead cells (identified by staining with LIVE/DEAD aqua amine; Invitrogen) were not included in the analysis.
5
Cytokine Profiling of Activated CD4 and CD8 T Cells
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Thawed CBMCs (1×106 cells/condition) were stimulated with phorbol 12-myristate 13-acetate (0.1 μg/mL) and ionomycin (1.0 μg/mL) or media alone (R10; 10% fetal bovine serum) for 5 h at 37 °C. Brefeldin A and monensin (BD Pharmingen) were added after 1 h of incubation at a final concentration of 10 mg/mL. After 5 h, cells were washed, fixed and permeabilized, as per the manufacturer’s instructions (FoxP3 Fix/Perm kit; eBiosciences), then stained with PE-Cy5.5 CD3 (Clone SK7), PE-e610 TNFα (clone MAB11, eBioscience), BV570 CD4 (Clone RPA-T4), BV711 CD8 (clone RPA-T8), BV650 CD45RA (clone HI100), BV605 CD45RO (clone UCHL1), BV510 γδTCR (clone B1), BV510 CD14 (Clone M5E2), BV510 CD19 (Clone HIB19), PE-Cy7 IFN-γ (Clone 4 S.B.3), BV421 IL-2 (clone MQ1-17H12), AF488 IL-8 (clone MQ1-17H12, Biolegend), and LIVE/DEAD aqua amine (Invitrogen). Data were collected on an LSR II (BD) and analyzed with FlowJo software (Treestar). Cord blood CD4 and CD8 T cell cytokine responses to mitogen stimulation were gated on non-naive effector populations by expression of CCR7 and CD45RO (as in Supplementary Fig. 1 ) for all except IL-8, which was gated on total CD4 T cells.
6
CFSE-based Proliferation Assay for Pf Schizont Extract
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The proliferative response to P. falciparum schizont extract (PfSE) was assessed by CFSE dilution. For subjects with sufficient CBMCs (n = 98), thawed CBMCs were rested for 1 h at 37 °C, washed in 10% Human AB (HAB) medium, and 3–4 × 106 cells were labeled with 1 μM CFSE (Molecular Probes) for 7 min. CFSE-labeled CBMCs were plated in 96-deep-well culture plates (Nunc) (1 × 106 cells/condition) and incubated with PfSE, uninfected red blood cells (uRBCs), or media alone. After 6 days, cells were washed twice in culture medium, then stained with PE-Cy5.5 CD3 (Clone SK7) eBioscience, BV570 CD4 (Clone RPA-T4), BV711 CD8 (clone RPA-T8), BV510 CD14 (Clone M5E2), BV510 CD19 (Clone HIB19) Biolegend, LIVE/DEAD aqua amine (Invitrogen) and counted.
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