NK cells were harvested and isolated from spleens of tumor bearing mice. Processed into single cell suspension following above protocol, and NK were isolated using an NK isolation kit (Stemcell) following manufacturer’s instructions. Isolated NKs were then stained using the following antibodies: APC-EpCAM, PE/Dazzle 594- NKp46 (Biolegend), FITC-Granzyme B (Biolegend). Nuclei were stained using 4′6-diamidino-2-phenylindole (DAPI). Following staining, cells were washed and resuspended in PBS and processed through Amnis Imagestream X Mk II imaging flow cytometer (Amnis, Seattle, WA). Analysis was performed using IDEAS 6.2 software (AMNIS, Seattle, WA).
Nk isolation kit
Manufactured by STEMCELL
The NK isolation kit is a laboratory product designed to isolate natural killer (NK) cells from biological samples. It provides a simple and efficient method to obtain purified NK cell populations for further analysis or experimentation.
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Lab products found in correlation
Interleukin 2 (il 2), by Thermo Fisher Scientific (2 mentions)
Epcam apc, by BioLegend (1 mentions)
Granzyme b fitc, by BioLegend (1 mentions)
Transwell migration chamber, by Corning (1 mentions)
Symphony a5, by BD (1 mentions)
High throughput sampler, by BD (1 mentions)
Live dead fixable near ir dead cell stain, by Thermo Fisher Scientific (1 mentions)
C34554, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by BD (1 mentions)
4 protocols using nk isolation kit
1
Isolation and Characterization of Tumor-infiltrating NK Cells
2
Transwell Assay for NK Cell Migration
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Cell migration was measured using a 24 well-format Transwell migration chamber (pore size, 5 µm, Corning, 3421). NK cells were isolated from splenocytes using Stem Cell Technologies NK isolation kit (19855) as described by manufacturer. First, NK cells were suspended in fresh medium (RPMI, 10% FBS, pen/strep) supplemented with 20 ng/mL IL-2 (PeproTech, 212-12) and seeded on 96-well plates (1 × 106 cells/well). After 24 h, cells were washed with PBS and suspended in migration buffer (RPMI, 0.2% FBS) at 1 × 106 cells/mL. For the migration to CCL5 chemokine, the migration buffer supplemented with 100 ng/mL CCL5 (PeproTech, 500-P118) was added in the lower compartment and 100 µl of cell suspension was added into each insert. After 4 h at 37 °C, the sample from lower compartment was collected and stained for NK1.1, NKp46 and Live/Dead marker (as described in Flow Cytometry) together with input sample. Samples were then suspended in equal volume and equal volume of sample was acquired using the BD High Throughput Sampler combined with BD Symphony A5 and the number of alive NKp46+, NK1.1+ positive cells per sample was analyzed. Migration rate was determined as a (nb of cells from sample / nb of cells from input sample) x 100%.
3
NK Cell Isolation and Proliferation Assay
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NK cells were isolated from splenocytes using Stem Cell Technologies NK isolation kit (19855) as described by manufacturer. Cells were washed twice with warm (37 °C) PBS to remove serum that affects staining. Then, cells were suspended in warm (37 °C) PBS at a concentration of 1 × 106 cells/ml and labelled with 5 mM of CFSE (ThermoFisher, C34554) at 37 °C for 15 min. Subsequently, cells were washed with warm (37 °C) PBS and then were suspended in fresh medium (RPMI, 10% FBS, pen/strep) supplemented with 20 ng/mL IL-2 (PeproTech, 212-12), seeded on 96-well plates (2 × 105 cells/well) and incubated for 120 h. After this time, cells were collected and stained with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain (ThermoFisher, L34975) (20 min, at 37 °C). Immediately after this step, cells were analyzed with flow cytometer (BD Symphony; BD Biosciences) with all necessary controls, i.e., CFSE negative, CFSE positive day 0 control.
4
Murine Splenocyte Isolation and Culture
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Spleens from mice were collected and placed in complete RPMI (Gibco#11875 RPMI 1640, 10% fetal calf serum, 1% penicillin/streptomycin) and minced through 100μm filter to create single cell suspensions. Splenocytes were then RBC lysed with lysis buffer (1M NH4NaCl, 1M HEPES) for 2 minutes and washed with complete RPMI. Splenocytes were counted and cultured at 10x10 6 cells per ml in complete RPMI, 10μM non-essential amino acids, 0.57μM 2-mercaptoethenol. When NK cell enrichment was used, NK cells were isolated from splenocytes using Stem Cell Technologies NK isolation kit (#19855) as described by manufacturer. Cytokines were obtained from Peprotech: IL-15 (210-15), IL-18 (B002-5), Pestka Biomedical Laboratories assay science IFNβ (12401-1) and used as indicated in figure legends. Splenocyte cultures were incubated at 37°C in a normal incubator (20% O2) or hypoxic chamber (1% O2). For intracellular cytokine measurements, NK cells were cultured for 20 hours then brefeldin A was added for the last four hours.
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