Splenic B cells were negatively selected by staining with biotinylated anti-CD43 (clone S7) and anti-CD11b (clone M1/70) antibodies for 20 minutes on ice, washing and incubating the cells with streptavidin beads (Dynabeads M-280 Invitrogen), following the manufacturer’s instructions. A Dynal Invitrogen Beads Separator was used to purify the cells. B1-8hi B cells were purified using biotinylated anti-CD43 (clone S7), anti-CD11b (clone M1/70) and anti-kappa (clone RMK-12) antibodies. OT-2 T cells from lymph nodes and spleen were purified using a mix of biotinylated antibodies: anti-B220 (clone RA3-6B2), anti-CD8 (clone 53-6.7), anti-NK1.1 (clone PK136), anti-CD11b, anti-GR1 (clone RB6-8C5), and anti-F4/80 (clone BM8). Splenic and lymph node B and T cells were maintained in RPMI 10% FBS supplemented with 2 mM l -glutamine, 100U/ml penicillin, 100 U/ml streptomycin, 20 µM β-mercaptoethanol, and 10 mM sodium pyruvate. Alternatively, plenic B cells and OT-2 T cells from lymph nodes were purified by negative selection using the CD43 (Ly-48) Microbeads (Miltenyi Biotec) and naive CD4 + T cell Isolation Kits (Miltenyi Biotec), respectively. The cell suspension was then loaded onto a MACS column and a MACS Separator was used to purify the cells. All antibodies used are listed in Supplementary Table 1 .
Naive cd4 t cell isolation kit
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
The Naive CD4+ T Cell Isolation Kit is a product that allows for the isolation of naive CD4+ T cells from human peripheral blood mononuclear cells (PBMCs). The kit uses a magnetic separation-based approach to negatively select for naive CD4+ T cells, leaving them untouched for further downstream applications.
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Lab products found in correlation
Tgf β, by R&D Systems (5 mentions)
Cd8 t cell isolation kit, by Miltenyi Biotec (5 mentions)
Ls column, by Miltenyi Biotec (5 mentions)
Rpmi 1640 medium, by Corning (4 mentions)
Naive cd8 t cell isolation kit, by Miltenyi Biotec (4 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (4 mentions)
Anti cd28, by BD (3 mentions)
Interleukin 2 (il 2), by R&D Systems (3 mentions)
Ionomycin, by Merck Group (3 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (3 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (3 mentions)
Anti ifn γ, by BD (2 mentions)
Anti il 4, by BD (2 mentions)
Il 12, by R&D Systems (2 mentions)
Anti cd44 biotin and anti biotin microbeads, by Miltenyi Biotec (2 mentions)
Anti pe microbeads, by Miltenyi Biotec (2 mentions)
Cd25 microbead kit, by Miltenyi Biotec (2 mentions)
Anti cd3 cd28 microbeads, by Thermo Fisher Scientific (2 mentions)
X vivo 15 medium, by Lonza (2 mentions)
Pan t cell isolation kit 2, by Miltenyi Biotec (2 mentions)
90 protocols using naive cd4 t cell isolation kit
1
Purification of Splenic B and T Cells
2
Isolation and Characterization of Murine Th2 Cells
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Spleens were harvested from mouse and were homogenized by passage through stainless steel mesh. Cells were suspended in Red Blood Cell Lysing Buffer, Hybri-Max TM (Sigma-Aldrich Japan, Tokyo, Japan). Next, naive CD4 + T cells were isolated from the cell suspensions using auto-MACS (Miltenyi Biotec K.K., Bergisch Gladbach, Germany) with Naive CD4 + T Cell Isolation Kits (Miltenyi Biotec K.K.) according to the manufacturer's instructions. Naive T cells were plated in 48-well cell culture plates at a final concentration of 1 × 10 6 cells/well using TexMACS Medium with CytoBox, Th2 Cell Kit (Miltenyi Biotec K.K.). Then, T cells were incubated with beads coated with CD3/CD28 antibody (T Cell Activation Expansion kit; Miltenyi Biotec K.K), LPA, LPA1 antagonists, and LPA2 antagonists for 24 h to evaluate LPA and its antagonist effect on Th2 differentiation. T cells were collected after LPA, LPA1 antagonist, and LPA2 antagonist stimulation and were used in flow cytometry studies. Supernatants were also harvested and stored at -80°C for the measurement of cytokines.
3
Naive CD4+ T Cell Polarization
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Naïve CD4+ T cells were purified from mouse spleen and MLNs using Naive CD4+ T Cell Isolation Kit (Miltenyi Biotec). Purified naïve CD4+ T cells were plated at a density of 2.5 × 105 cells/well at 37 °C in 48-well plates pre-coated with anti-CD3e (Clone: 145-2C11, 1 μg/ml, BD Biosciences) and anti-CD28 (Clone: 37.51, 1 μg/ml, BD Biosciences) antibodies. Activated cells were polarized under Th0 (no supplement), Th1 [IL-12 (5 ng/ml, R&D Systems), IL-2 (10 ng/ml, R&D Systems) and anti-IL-4 antibody (5 μg/ml, BD Biosciences)], Treg [TGF-β (5 ng/ml, R&D Systems), IL-2 (10 ng/ml, R&D Systems), anti-IFN-γ (5 μg/ml, BD Biosciences), anti-IL-12 p40/p70 (5 μg/ml, BD Biosciences) and anti-IL-4 (5 μg/ml, BD Biosciences) antibodies] differentiation conditions in complete RPMI medium or polarized in splenic DC-conditioned medium (overnight culture supernatant of CpG-ODN-stimulated splenic DCs).
4
Modulation of Naive CD4+ T Cells by CNS APCs
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Naïve CD4+ T cells were isolated from splenocytes and lymph node cells with the naive CD4+ T cell isolation kit (Miltenyi Biotec). To obtain CNS APCs, brains from newborn mice were collected, dissociated with scissors and digested with collagenase type 4 (2 mg/ml, Worthington Biochemical) for 1 hour at 37 °C. Mononuclear cells in the interphase between 30% and 70% of Percoll (GE healthcare) were isolated by density-cut centrifugation as described38 . These cells were cultured for 4 days and then activated by TNF-α (10 ng/ml, BioLegend) and IL-17 (25 ng/ml, BioLegend) in the presence and absence of SCFAs (C2 at 10 mM, C3 at 1 mM, C4 at 0.5 mM) for additional 3 days in DMEM medium (10% FBS). Adherent glial cells, largely composed of microglial cells, were co-cultured as APCs with naïve CD4+ T cells at 1:10 ratio for 5–6 days in the presence of Staphylococcal enterotoxin B (SEB, 5 μg/ml, List Labs) to stimulate naïve CD4+ T cells isolated from the spleen and lymph nodes of unimmunized C57BL/6 mice. For flow cytometry of cytokine expression by T cells, total draining LN cells from MOG-immunized mice were cultured with MOG35–55 peptide (40 μg/ml) and hIL-2 (100 U/ml) in the presence or absence of SCFAs (C2, 10 mM; C3, 1 mM; C4, 0.5 mM) for 5 days.
5
Isolation of Naive CD4+ T-Cells
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Autologous naive T-cells were separated from human blood mononuclear cells using the naive CD4+ T-cell isolation kit based on negative selection according to the manufacturer's instruction (Miltenyi Biotec).
6
Adoptive Transfer of SMARTA and Memory CD8 T Cells
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For adoptive transfer of SMARTA CD4 T cells, naive CD4 T cells were negatively isolated from the spleen of wild-type SMARTA mice (CD45.1+) using a naive CD4 T cell isolation kit (Miltenyi Biotec). 104 naive SMARTA CD4 T cells were then adoptively transferred i.v. into WT or miR-181a−/− hosts (CD45.2+) before LCMV infection. For adoptive co-transfer experiments of WT and miR-181a−/− SMARTA cells, naive CD4 T cells were isolated from the spleens of WT (CD45.1+) and miR-181a−/−(CD45.2+) SMARTA mice. Cells were then mixed at a 1:1 ratio, and a total of 1×104 YFP+ SMARTA CD4 T cells were injected i.v. into B6 recipient mice 1 day prior to LCMV infection. For recall response of memory CD8 T cells, WT and miR-181a−/− mice were infected with LCMV. On day 50, CD8 T cells were negatively isolated from the spleen using a CD8 T cell isolation kit (Miltenyi Biotec), and CD44+ memory CD8 T cells were then positively enriched using anti-CD44-biotin and anti-biotin microbeads (Miltenyi Biotech). ~8×104 YFP+ CD44+ GP33-speicifc memory CD8 T cells from WT and miR-181a−/− mice were transferred into B6 hosts, followed by LCMV infection one day later.
7
Eosinophil Migration Assay with Tfh or Naive CD4+ T Cells
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FACS‐sorted Tfh cells or MACS‐sorted naïve CD4+ T cells by a Naive CD4+ T Cell Isolation Kit (Miltenyi Biotec) were placed in the bottom of tissue culture wells in the presence or absence of PMA (25 ng/mL; Sigma‐Aldrich) and ionomycin (1 μg/mL; Sigma‐Aldrich). Eosinophils from the liver were incubated with anti‐PE microbeads (Miltenyi Biotec) after staining with Siglec‐F‐PE (clone E50‐2440, BD Pharmingen) and purified by MACS. Then, transwell inserts with MACS‐purified eosinophils (>95% pure) were placed on top. All of the transwell experiments were repeated three times with 3 wells for each treatment. The percentage of migrated eosinophils in each group was determined using a FACSVerse cytometer (BD Biosciences).
8
Treg Cell Differentiation Assay
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For DC-T cell co-cultures, naive CD4+ T cells were isolated from the spleens of Foxp3GFP mice via negative selection of magnetically labeled cells according to the manufacturer’s instructions (Naive CD4+ T Cell Isolation Kit, Miltenyi Biotec). CD4+ T cells were then co-cultured with CD11c+ DCs isolated from Smad7fl/fl and DC-Smad7−/− mice at a 1:3 DC:T cell ratio in U-bottom 96-well plates. TGF-β (0.5 ng/mL) was added to Treg-polarizing conditions immediately upon setting DC-T cell co-cultures. All cells were cultured in X-VIVO serum-free media.
9
Antigen-Specific T Cell Proliferation Assay
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The sorted BMDCs were cultured and treated as per the above method. Finally, the prepared cells were pulsed with OVA323-339 for 1 h. Meanwhile, splenic cells were isolated from female OT-II mice (6–8 weeks). After a grinding step, red blood cell lysis buffer was added to remove the red blood cells; then, the cells were washed twice with cold PBS to prepare the single-cell suspension. The naïve CD4+ T cells were purified using a Naive CD4+ T Cell Isolation Kit (Miltenyi), according to the manufacturer's instructions. Subsequently, the purified cells were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen) following the standard protocols, and then co-cultured with OVA323-339 pulsed BMDCs in a 24-well plate for stimulating antigen-specific T cell proliferation. The ratios of DCs: T was 1:10, 1:30, and 1:100, the number of T cells was the same in each groups, with a proportional change in the number of DCs. After 5 days, the cells were collected, stained with CD4-PE, and detected using a flow cytometer.
10
Adoptive Transfer of Naive CD4+ T Cells
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Naive CD4+ T cells were enriched from 6- to 8-week-old CKO and control mice using a Naive CD4 T Cell Isolation Kit (130-104-453, Miltenyi Biotec). Enriched cells were counted, and 5 × 106 cells were adoptively transferred into NOD/SCID recipient mice by intraperitoneal injection. The recipient mice were weighed immediately after T cell transfer and then twice a week thereafter. Their clinical signs were assessed by monitoring weight loss and change in stool consistency.
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