To obtain streptavidin-PE-conjugated tetramer, biotinylated Dd/NP166–174 monomeric protein was purified and tetramerized at an 8:1 molar ratio with streptavidin-PE (Invitrogen, San Diego, CA, USA) according to the manufacturer's protocol. At day 10 post challenge, each group of mice was sacrificed and tracheotomy was performed. The lungs were perfused with 5 mL of PBS with 10 U/mL heparin (Sigma) using a syringe with a 25-gauge needle. The lung tissues were homogenized with 3 mL of Iscove's Modified Dulbecco's Medium (IMDM) through 70-µm cell strainers to obtain single-cell suspensions. Centrifugation was conducted afterward, lymphocytes were resuspended in fresh IMDM and washed with fluorescence-activated cell sorting (FACS) buffer (0.5% FBS and 0.09% NaN_3 in PBS). After blocking with anti-mouse CD16/CD32 (BD Biosciences, Franklin Lakes, NJ, USA), cells were stained with anti-CD8a APC (clone 53-6.7, Biolegend, San Diego, CA, USA), anti-CD44 FITC (clone IM7, Biolegend), and Dd/NP(I) tetramer-PE or Dd/NP(V) tetramer-PE. After staining, the cells were washed and fixed with FACS lysing solution for 15 minutes at RT in the dark. Cells were washed with FACS buffer twice and stored at 4℃ in the dark until FACS analysis.
Anti cd44 fitc clone im7
Manufactured by BioLegend
Sourced in United States
Anti-CD44 FITC (clone IM7) is a fluorescent-labeled monoclonal antibody that binds to the CD44 antigen. CD44 is a cell surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by Thermo Fisher Scientific (2 mentions)
Moflo facs sorter, by Agilent Technologies (2 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (2 mentions)
Pe anti cd62l clone mel 14, 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)
Heparin, by Merck Group (1 mentions)
Streptavidin pe, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd16 cd32, by BD (1 mentions)
Anti cd8a apc clone 53 6, by BioLegend (1 mentions)
Facscalibur, by BD (1 mentions)
Brilliant staining buffer, by BD (1 mentions)
Celesta flow cytometer, by BD (1 mentions)
Fvs510, by BD (1 mentions)
Polyclonal rat igg, by Merck Group (1 mentions)
Anti cd44 fitc, by BD (1 mentions)
Anti cd8 pe cy5 clone 53 6.7, by BioLegend (1 mentions)
Anti cd45 apc clone 30 f11, by BioLegend (1 mentions)
7 protocols using anti cd44 fitc clone im7
1
Quantifying Antigen-Specific CD8+ T Cells
2
Cytokine-Producing Cell Analysis in Lung Lymphocytes
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For cytokine-producing cell analysis, 1×106 lung lymphocytes resuspended in IMDM with 10% FBS were prepared. As a positive control, cells were treated with 50 µg/mL phorbol 12-myristate 13-acetate (1:1,000) and 500 µg/mL ionomycin (1:1,000). NP-specific stimulation was conducted with 10 µM B-NP(I) peptide (FSPIRI TFL) or B-NP(V) peptide (FSPIRV TFL). The cells were also treated with 10 ng of recombinant human IL-2 protein (Biolegend) and Brefeldin A (1:1,000, eBioscience, San Diego, CA, USA), and were incubated for 5 hours at 37℃. Stimulated cells were blocked with anti-mouse CD16/32 for 10 minutes at RT and stained with anti-CD8a PE (clone 53-6.7, Biolegend) and anti-CD44 FITC (clone IM7, Biolegend) for 30 minutes at 4℃ in the dark. After permeabilization, intracellular cytokines were stained with anti-interferon-γ (IFN-γ) antibody (clone XMG 1.2, Biolegend) for 30 minutes at RT in the dark. The cells were washed twice and stored at 4℃ in the dark until FACS analysis. All stained cells were analyzed using FACSCalibur (BD Biosciences), and data were analyzed with FlowJo software (TreeStar Inc., Ashland, OR, USA).
3
Multicolor Flow Cytometry Staining Protocol
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Cells were washed in PBS and stained for viability using FVS510 (BD Biosciences) which was diluted 1:1,000 in PBS and 100 µL was added to each sample of cells and left in the dark at room temperature for 15 min and then washed twice in FACs buffer (PBS containing 0.5% BSA, 5 µM EDTA and 0.5% sodium azide). To reduce nonspecific binding, samples were incubated with 50 µL of polyclonal rat IgG (Sigma) (diluted 1:50 in FACs buffer) for 15 min on ice and protected from light, then washed once in FACs buffer by spinning at 400 g for 5 min and removing supernatant. Samples were then stained for flow cytometry by adding 50 µL of anti-CD44-FITC (clone IM7 at 1/200; Biolegend) in Brilliant Staining Buffer (BD Biosciences). Staining with TGMs used Alexa fluor-labeled proteins made as described above. TGMs labeled with AF488 and AF594 were used at a final concentration of 5 μg/mL. Samples that were also stained for intracellular antigens were fixed and permeabilized using Foxp3 Transcription Factor Buffer kit (Invitrogen, USA) and stained with anti-Foxp3-ef450 (clone FJK-16s, eBioscience, San Diego, California, USA, 1/100) in perm/wash buffer. Samples were then washed prior to analysis on a BD Celesta flow cytometer (BD Biosciences).
4
Characterization of Antigen-Specific CD8+ T Cells
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For tetramer staining, cell suspensions obtained from peripheral blood or lung tissue were blocked with anti-mouse CD16/32 and stained with anti-CD44 FITC (clone IM7; BioLegend, San Diego, CA), anti-CD8 PE-Cy5 (clone 53–6.7; BioLegend), anti-CD45 APC (clone 30-F11; BioLegend) and MHC class I tetramer HA533-541 (H-2Kd/IYSTVASSL).
For intracellular staining, the cell suspensions were incubated for 6 hours with HA533-541 peptide/recombinant human IL-2 (BioLegend) or phorbol myristate acetate (PMA)/ionomycin in Iscove’s Modified Dulbecco’s Media containing 10% FBS and Brefeldin A (eBioscience). Following incubation, the cells were blocked with anti-mouse CD16/32, surface stained with anti-CD44 FITC, anti-CD8 PE-Cy7 and anti-CD45 APC, and fixed in BD FACS lysing solution (BD Pharmingen). The fixed samples were permeabilized in FACS buffer containing 0.5% saponin and stained with anti-IFN-γ PE (clone XMG1.2; eBioscience).
For intracellular staining, the cell suspensions were incubated for 6 hours with HA533-541 peptide/recombinant human IL-2 (BioLegend) or phorbol myristate acetate (PMA)/ionomycin in Iscove’s Modified Dulbecco’s Media containing 10% FBS and Brefeldin A (eBioscience). Following incubation, the cells were blocked with anti-mouse CD16/32, surface stained with anti-CD44 FITC, anti-CD8 PE-Cy7 and anti-CD45 APC, and fixed in BD FACS lysing solution (BD Pharmingen). The fixed samples were permeabilized in FACS buffer containing 0.5% saponin and stained with anti-IFN-γ PE (clone XMG1.2; eBioscience).
5
Isolation and Characterization of Salivary Gland Leukocytes
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Leukocytes were isolated directly ex vivo from the SGs of 10BiT mice on day 14 p.i. (minimum n = 5 mice per group with three replicates). Pooled cells were labeled using a Zombie Aqua Fixable Viability Kit (BioLegend) and stained with anti-CD16/CD32 (Fc block, BioLegend) followed by anti-CD4–BV605 (clone RM4-5, BioLegend), anti-CD44–FITC (clone IM7, BioLegend), anti-CD62L–PE-Cy7 (clone MEL-14, BioLegend), and anti-CD90/90.1–PE (clone OX-7, BioLegend). Cells were sorted as CD4+ CD44+ CD62L− CD90/90.1+ (Thy1.1+) or CD90/90.1− (Thy1.1−) populations directly into Buffer RLT or Buffer RLT Plus (QIAGEN) using a modified FACS Aria II (BD Biosciences). Total RNA was extracted using an RNeasy Micro Kit or an RNeasy Mini Kit (QIAGEN), and RNA integrity scores were determined using an RNA 6000 Pico Kit (Agilent).
6
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.
7
Isolation and Characterization of Memory Th17 Cells
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