Human CD4+ T cells were isolated from buffy coats of healthy donors (obtained from Blodbanken, Oslo, Norway) using the Dynabeads CD4 Positive Isolation Kit (Life Technologies AS, Norway) according to the manufacturer's protocol. A conventional carboxyfluorescein diacetate succinimidyl ester (CFSE)‐based T‐cell proliferation was performed to test the antiproliferative effect of C19 and BPTES. In brief, CFSE (2.5 µm )‐labelled human CD4+ T cells (1 × 106 cells·well−1) were incubated with C19 (25 µm ) and BPTES (25 µm ) followed by stimulation with anti‐CD3/CD28 beads (1 : 1; beads: cells ratio) (Dynabeads; Invitrogen, Waltham, MA, USA) for 96 h. After 4 days of incubation, beads were removed; cells were washed with 1x PBS, and T‐cell proliferation was assessed by measuring the CFSE dilution using flow cytometry.
Dynabeads cd4 positive isolation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Norway
The Dynabeads CD4 Positive Isolation Kit is a laboratory product designed for the isolation of CD4-positive cells from biological samples. The kit utilizes magnetic bead technology to selectively capture and isolate CD4-positive cells, which are a type of T lymphocyte that plays a crucial role in the immune system.
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Lab products found in correlation
Polyethylenimine (pei), by Polysciences (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (2 mentions)
Dynabeads cd8 positive isolation kit, by Thermo Fisher Scientific (2 mentions)
Aria 2, by BD (2 mentions)
Easysep mouse na ve cd4 t cell kit, by STEMCELL (2 mentions)
Dynabeads pan mouse igg, by Thermo Fisher Scientific (2 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Glutamine, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Allprep dna rna protein mini kit, by Qiagen (1 mentions)
Lsrfortessa sorp cytometer, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Facs software, by BD (1 mentions)
Geneart crispr nuclease vector, by Thermo Fisher Scientific (1 mentions)
Geneart genomic cleavage detection kit, by Thermo Fisher Scientific (1 mentions)
32 protocols using dynabeads cd4 positive isolation kit
1
Antiproliferative Effect of C19 and BPTES on CD4+ T Cells
2
Transfection and CD4+ Cell Isolation
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Twenty-four hours after seeding in 10 cm dishes, HEK293T and HeLa cells were transfected with 10 μg of DNA using polyethylenimine (PEI, Polysciences) as transfection vehicle with a PEI to DNA w/w ratio of 4/1 and 2/1, respectively. To increase the number of transfected cells in the cell cultures, CD4 positive cells were isolated before the evaluation of PINK1 and parkin protein levels. For this purpose, 72 h after seeding, cells were subjected to CD4 isolation with the Dynabeads® CD4 Positive Isolation Kit (Life Technologies), according to manufacturer’s instructions. Isolated CD4 positive cells were then plated into six well plates for western blot analysis.
3
Isolation and Activation of Human CD4+ T Cells
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Human CD4+ T cells were isolated from buffy coats of healthy donors (supplied by Blodbanken, Oslo, Norway) using the Dynabeads CD4 Positive Isolation kit (Life Technologies AS, Norway). Buffy coats were diluted with RPMI 1640 (1:2) and 1ml EDTA and rotated for 15min at 4°C. The kit was used to acquire CD4+ cells according to the supplied protocol. Five million cells/ml were maintained in RPMI 1640 (Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% heat inactivated FBS (Sigma Aldrich); 2mM glutamine (Sigma Aldrich) 0.5% Penicillin-Streptomycin (Sigma Aldrich) unless stated otherwise. CD4+ T stimulation was initiated by adding washed Human T-Activator CD3/CD28 Dynabeads at a 1:4 bead/cells ratio (Life Technologies). CD4+ T cells were stimulated in this fashion during the entire study up to indicated time points.
4
Isolation and Maintenance of CD4+ Lymphocytes
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PBMCs were accumulated from randomized leukocyte concentrates obtained from apheresis products of anonymized healthy adult donors from the university hospital’s Department of Immunohematology and Transfusion Medicine, University of Wuerzburg, as described previously [45 (link)]. This study has been approved by the Ethic Committee of the Medical Faculty of Wuerzburg. Leukocytes were isolated using Ficoll-Paque (LINARIS Biologische Produkte GmbH, Dossenheim, Germany) for 25 min at 530×g. CD4+ lymphocytes were further enriched by positive isolation using the Dynabeads® CD4 Positive Isolation Kit (Life Technologies, Carlsbad, CA) according to the manufacturers’ instructions. The purity of the isolated CD4+ lymphocytes was > 97%. CD4+ cells were maintained in RPMI 1694 and DMEM (Sigma-Aldrich), respectively, supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Life Technologies, Carlsbad, CA). Incubation was carried out at 37°C in a humidified atmosphere with 5% CO2.
5
Isolation and Activation of Human CD4+ T Cells
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Human CD4+ T cells were isolated from buffy coats of healthy donors (supplied by Blodbanken, Oslo, Norway) using the Dynabeads CD4 Positive Isolation kit (Life Technologies AS, Oslo Norway) as previously described [22 (link)]. Buffy coats were diluted 1 : 2 with RPMI 1640 (Sigma–Aldrich, St. Louis, MO, U.S.A.) and 1 ml EDTA and rotated for 15 min at 4°C. CD4+ T cells were then isolated according to the company protocol. Cells were maintained at 5 × 106 cells/ml unless otherwise stated. CD4+ T-cell activation was initiated by adding pre-washed Human T-Activator CD3/CD28 Dynabeads at a 1 : 4 bead/cells ratio (Life Technologies).
6
T-Cell Enrichment from PBMC and Tumor
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The single cell suspensions of both PBMC and whole tumor were enriched for T-cells through use of Dynabeads CD4 Positive Isolation Kit (Life Technologies, Cat # 11331D) and Dynabeads CD8 Positive Isolation Kit (Life Technologies, Cat # 11333D). These kits were used in parallel to isolate t-cells as opposed to a CD3 + T-cell positive isolation kit to prevent accidental activation of these cells through engagement of the CD3 receptor. Cells were prepared in accordance with the kit protocol provided.
7
Isolation and Extraction of CD4+ T Cell Biomolecules
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Peripheral blood mononuclear cells were obtained from each patient. Subsequently, CD4+ T cells were isolated using the Dynabeads CD4 positive Isolation Kit from Life Technologies, following the manufacturer’s instructions. The purity of the cell fraction was > 98%, as determined by flow cytometry (BD FACSCalibur). Finally, the extraction of RNA and DNA from the CD4+ cells was performed using the AllPrep DNA/RNA/Protein MiniKit (Qiagen). The obtained RNA and DNA were quantified by spectrophotometry using a NanoDrop, purity was also assessed by spectrophotometry, and quality of nucleic acids was analyzed by electrophoresis. The RNA and DNA were conserved at − 80 °C until their further use.
8
Isolation and Characterization of Naive CD4+ T Cells
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Naive CD62L+CD44low CD4+ T cells were enriched by positive selection (Dynabeads CD4 Positive Isolation Kit; Invitrogen); labeled with fluorescent antibodies specific for CD4, CD8, CD44, and CD62L; and purified by sorting on a BD INFLUX (Becton Dickinson) with BDFACS software, version 1.2.0.142 (BD). Cells for flow cytometry were resuspended in PBS with 1% FBS, 2 mM EDTA, 0.01% azide, and then incubated in anti-(a)CD16/CD32 followed by the appropriate fluorescent antibodies and DAPI staining to exclude nonviable cells (Table S1 ). Data were acquired using a custom LSR-Fortessa SORP cytometer (Becton Dickinson) with BD FACSDiva software, version 6.1.1 (BD), and analyzed using FlowJo software (Tree Star).
9
Isolation and analysis of YFP-labeled CD4 T cells from mouse tissues
10
Isolation and analysis of YFP-labeled CD4 T cells from mouse tissues
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For analysis of YFP-labeled CD4 T cells in Tbx21RFP-CreERT2 mice, CD4 T cells in spleens and lymph nodes were enriched using the Dynabeads CD4 Positive Isolation Kit (Invitrogen). To isolate lymphocytes from tissues, mice were euthanized and immediately perfused with 20 mL PBS. Small and large intestines were removed, flushed with PBS and Peyer’s patches were removed. 0.5 cm-long fragments of intestines were washed in PBS and incubated in PBS supplemented with 5% fetal calf serum, 1% L-glutamine, 1% penicillin-streptomycin, 10 mM HEPES, 1 mM dithiothreitol, and 1 mM EDTA for 15 minutes. Samples were washed and incubated in digest solution (RPMI supplemented with 5% fetal calf serum, 1% L-glutamine, 1% penicillin-streptomycin, 10 mM HEPES, 1 mg/mL collagenase, and 1 U/mL DNase I) for 10 minutes twice. After filtering through a 100-μm strainer, cells were resuspended in 35% Percoll to eliminate debis. Lymphocytes from livers and lungs were isolated by 50–60 min incubation in digest solution, filtered through 100-μm strainers, and after debris removal in 35% Percoll, purified by centrifugation (1000×g, 7.5 min) over a step-wise 44%/67% Percoll gradient at room temperature.
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