rosettesep nk cell enrichment kit  (STEMCELL Technologies Inc)

Journal: STAR Protocols

Article Title: Isolation, expansion, and adoptive transfer of human ILC2s for the treatment of mice bearing liquid and solid tumors

doi: 10.1016/j.xpro.2024.103096

Figure Lengend Snippet: The procedure for isolating total ILCs 1. Pool blood into a 50 mL tube. 2. Dilute blood with DPBS in a 1:1 ratio and then add 500 μL RosetteSep Human NK Cell Enrichment Cocktail. Incubate the cocktail with blood on the Fisherbrand Platform Rotator (as per steps 1 and 2 in the text). 3. Add 20 mL Ficoll-Paque to a 50 mL conical tube and layer the blood mixture on top of Ficoll-Paque (as per step 3 in the text). 4. Perform density gradient centrifugation (as per step 4 in the text). 5. Collect fractions (blue arrow: enriched NK cells) from the 50 mL tube (as per steps 5 and 6 in the text). 6. Add red blood cell (RBC) lysis for 5 min (as per steps 7 and 8 in the text). 7. Perform enrichment of ILCs and isolate total ILCs (as per steps 9–16 in the text).

Article Snippet: RosetteSep human NK cell enrichment kit , STEMCELL , Cat# 15065.

Techniques: Gradient Centrifugation, Red Blood Cell Lysis

Journal: STAR Protocols

Article Title: Isolation, expansion, and adoptive transfer of human ILC2s for the treatment of mice bearing liquid and solid tumors

doi: 10.1016/j.xpro.2024.103096

Figure Lengend Snippet:

Article Snippet: RosetteSep human NK cell enrichment kit , STEMCELL , Cat# 15065.

Techniques: Recombinant, Red Blood Cell Lysis, Software, Cell Culture, Sterility, Saline, Ointment

Journal: Cells

Article Title: A Critical Role of Culture Medium Selection in Maximizing the Purity and Expansion of Natural Killer Cells

doi: 10.3390/cells13131148

Figure Lengend Snippet: Freshly isolated PBMCs were cultured in different media and adherently selected for NK cell enrichment. ( A ) Schematic illustration of the adherent selection process for the enrichment of NK cells: the culture begins with PBMCs, which are activated overnight to enable the selection of adherent cells on the next day. Then, the adherent cells are further cultured. Flow cytometry analysis was performed with the PBMCs before seeding, the cells that were removed during the adherent selection process, and the adherently selected and cultivated cells. ( B ) The number of NK cells left after the adherent selection process. ( C ) The percentage of CD56 + /CD3 − NK cells in freshly isolated PBMCs and achieved purity of adherently selected cell cultures of different donors after culture in PRIME-XV NK Cell CDM (CDM) or NK MACS ® (NK MACS), as determined by flow cytometry.

Article Snippet: For the isolation of NK cells, the RosetteSep™ human NK cell enrichment kit (Stemcell Technologies Germany GmbH, Cologne, Germany) was used according to the manufacturer’s instructions.

Techniques: Isolation, Cell Culture, Selection, Flow Cytometry

Journal: Cells

Article Title: A Critical Role of Culture Medium Selection in Maximizing the Purity and Expansion of Natural Killer Cells

doi: 10.3390/cells13131148

Figure Lengend Snippet: Freshly isolated PBMCs and RosetteSep™ human NK cell enrichment kit purified NK cells were cultured in NK MACS ® medium supplemented with human AB serum and 500 U/mL IL-2 for 20 days. For the isolation of NK cells, untreated (R) and two-fold concentrated (RC) buffy coats were used. ( A ) Schematic representation illustrating the NK cell purification process utilizing the RosetteSep™ cocktail. The cocktail comprises tetrameric antibodies that bind red blood cells to non-NK cells, facilitating the separation of NK cells through density gradient centrifugation. ( B ) Median with the interquartile range of the number of NK cells that were purified under the application of the RosetteSep™ human NK cell enrichment kit using untreated buffy coats ( n = 14). ( C ) Percentage of NK cells after isolation and after 20 days of cultivation ( n = 2; mean ± SEM). ( D ) NK cell expansion after 20 days of cultivation ( n = 2; mean ± SEM). ( E ) Number of NK cells after 20 days of cultivation ( n = 2; mean ± SEM).

Article Snippet: For the isolation of NK cells, the RosetteSep™ human NK cell enrichment kit (Stemcell Technologies Germany GmbH, Cologne, Germany) was used according to the manufacturer’s instructions.

Techniques: Isolation, Purification, Cell Culture, Gradient Centrifugation

Journal: Cells

Article Title: A Critical Role of Culture Medium Selection in Maximizing the Purity and Expansion of Natural Killer Cells

doi: 10.3390/cells13131148

Figure Lengend Snippet: Freshly isolated NK cells were cultured in NK MACS ® medium supplemented with IL-2 (500 U/mL) and either FCS, human AB serum, human pooled serum from different blood types, MultiPL’100 or Panexin CD (see figure legend; n = 3; mean ± SEM). In one of the three assays performed, NK cells isolated from two donors were mixed. Only living cells were counted to determine the expansion. ( A ) NK cell expansion during the cultivation period using different sera or serum replacements. ( B ) NK cell expansion after three weeks of cultivation using different sera or serum replacements. The statistical analysis was performed per one-way ANOVA, comparing AB serum with every other group, applying Dunnett’s correction for multiple comparisons (ns = not significant; * p < 0.0332).

Article Snippet: For the isolation of NK cells, the RosetteSep™ human NK cell enrichment kit (Stemcell Technologies Germany GmbH, Cologne, Germany) was used according to the manufacturer’s instructions.

Techniques: Isolation, Cell Culture

Journal: Cells

Article Title: A Critical Role of Culture Medium Selection in Maximizing the Purity and Expansion of Natural Killer Cells

doi: 10.3390/cells13131148

Figure Lengend Snippet: Freshly isolated NK cells were cultured in NK MACS ® medium supplemented with human AB serum with different stimulatory factors and their proliferation, as well as cytotoxic potential were determined. ( A ) Arrows pointing at activation/expansion beads (A/E Beads), microscopic image (400×). ( B ) Expansion of NK cells cultured with A/E Beads and IL-2 (500 U/mL), with either A/E Beads or IL-2 (500 U/mL) only or without stimulatory factors. ( C ) Expansion of NK cells stimulated with the cytokines IL-2 (500 U/mL), IL-15 (100 U/mL), or IL-21 (1 U/mL) as well as with the A/E Beads for 10 days in different combinations. ( D ) Specific lysis of target cells determined in a killing assay performed with the NK cells shown in C as effector cells and K562 cells as target cells (effector:target = 1:1). The symbols represent technical replicates and the bars represent the mean ± SD (ns = not significant; * p < 0.0332; *** p < 0.002). The statistical analysis was performed per one-way ANOVA, comparing each group with every other group, and applying Tukey correction for multiple comparisons; however, only selected comparisons are indicated.

Article Snippet: For the isolation of NK cells, the RosetteSep™ human NK cell enrichment kit (Stemcell Technologies Germany GmbH, Cologne, Germany) was used according to the manufacturer’s instructions.

Techniques: Isolation, Cell Culture, Activation Assay, Lysis

Journal: Cells

Article Title: A Critical Role of Culture Medium Selection in Maximizing the Purity and Expansion of Natural Killer Cells

doi: 10.3390/cells13131148

Figure Lengend Snippet: Freshly isolated PBMCs and NK cells were cultured in NK MACS ® medium supplemented with human AB serum and 500 U/mL IL-2 for 21 days. ( A ) Percentage of NK cells cultured in the presence of other PBMCs and in a RosetteSep™ human NK cell enrichment kit purified culture ( n = 3; mean ± SEM). ( B ) NK cell expansion fold in the PBMC culture (left) and the RosetteSep™ human NK cell enrichment kit purified NK cell culture (right) ( n = 3; mean ± SEM).

Article Snippet: For the isolation of NK cells, the RosetteSep™ human NK cell enrichment kit (Stemcell Technologies Germany GmbH, Cologne, Germany) was used according to the manufacturer’s instructions.

Techniques: Isolation, Cell Culture, Purification

Journal: Cells

Article Title: A Critical Role of Culture Medium Selection in Maximizing the Purity and Expansion of Natural Killer Cells

doi: 10.3390/cells13131148

Figure Lengend Snippet: Freshly isolated PBMCs were cultured in NK MACS ® medium supplemented with human AB serum and 500 U/mL IL-2 for 21 days ( n = 3). During the cultivation periods, the receptor expression levels of specific NK cell activation markers were determined through flow cytometric analysis. ( A ) Schematic representation of the expression levels of NK cell activating receptors in the co-presence of other PBMCs. ( B ) Expression levels of the NK cell activation marker CD16. ( C ) Expression levels of the NK cell activation marker NKp46. ( D ) Expression levels of the NK cell activation marker NKG2D. ( E ) Expression levels of the NK cell activation marker I-CAM1.

Article Snippet: For the isolation of NK cells, the RosetteSep™ human NK cell enrichment kit (Stemcell Technologies Germany GmbH, Cologne, Germany) was used according to the manufacturer’s instructions.

Techniques: Isolation, Cell Culture, Expressing, Activation Assay, Marker

Journal: Cells

Article Title: A Critical Role of Culture Medium Selection in Maximizing the Purity and Expansion of Natural Killer Cells

doi: 10.3390/cells13131148

Figure Lengend Snippet: Freshly isolated and purified NK cells were cultured in NK MACS ® medium supplemented with human AB serum and 500 U/mL IL-2 for 21 days ( n = 3). During the cultivation periods, the receptor expression levels of specific NK cell activation markers were determined through flow cytometric analysis. ( A ) Schematic representation of the expression levels of NK cell activating receptors in the absence of other PBMCs. ( B ) Expression levels of the NK cell activation marker CD16. ( C ) Expression levels of the NK cell activation marker NKp46. ( D ) Expression levels of the NK cell activation marker NKG2D. ( E ) Expression levels of the NK cell activation marker I-CAM1.

Article Snippet: For the isolation of NK cells, the RosetteSep™ human NK cell enrichment kit (Stemcell Technologies Germany GmbH, Cologne, Germany) was used according to the manufacturer’s instructions.

Techniques: Isolation, Purification, Cell Culture, Expressing, Activation Assay, Marker

Journal: Cells

Article Title: A Critical Role of Culture Medium Selection in Maximizing the Purity and Expansion of Natural Killer Cells

doi: 10.3390/cells13131148

Figure Lengend Snippet: NK cells cultivated in NK MACS ® medium supplemented with human AB serum and IL-2 potently kill primary glioblastoma cancer stem cells from different donors. Adapted from . ( A ) Glioblastoma tissue samples were minced and incubated with proteolytic enzymes and cultured under application by serial trypsinization to generate a cancer stem cell fraction. ( B ) Specific lysis of target cells determined in a killing assay performed with the activated NK cells as effector cells and glioblastoma cancer stem cells as target cells (effector:target = 1:1, 1:3, 1:9). The symbols represent technical replicates and the bars represent the mean ± SD.

Article Snippet: For the isolation of NK cells, the RosetteSep™ human NK cell enrichment kit (Stemcell Technologies Germany GmbH, Cologne, Germany) was used according to the manufacturer’s instructions.

Techniques: Incubation, Cell Culture, Lysis