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StemSpan™ NK细胞分化添加物（100X）

用于淋系祖细胞向NK细胞分化的添加物
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产品号 #09950_C

用于淋系祖细胞向NK细胞分化的添加物

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Products for Your Protocol
To see all required products for your protocol, please consult the Protocols and Documentation.
  1. StemSpan™ SFEM II
    StemSpan™ SFEM II

    Serum-free medium for culture and expansion of hematopoietic cells

  2. UM729
    UM729

    Pyrimido-indole derivative that enhances HSC self-renewal in vitro

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概述

StemSpan™ NK细胞分化添加物（100X）能支持淋系祖细胞向自然杀伤（NK）细胞分化。

该添加物是StemSpan™ NK细胞生成试剂盒或STEMdiff™ NK细胞分化试剂盒的组分之一，也可单独购买以方便您的使用。可使用该添加物将CD34+细胞在含StemSpan™淋系祖细胞扩增添加物（10X）的StemSpan™ SFEM II培养基中、在StemSpan™淋系分化包板材料（100X）包被的培养板上分化产生的淋系祖细胞进一步分化为NK细胞。

StemSpan™NK细胞分化添加物（100X）需与StemSpan™ SFEM II和UM729搭配使用以促进向CD56+ NK细胞的分化。

关于NK细胞生成方法的详细信息，请参阅技术手册。

Subtype
Supplements
 
Cell Type
Hematopoietic Stem and Progenitor Cells, NK Cells
 
Species
Human
 
Application
Differentiation
 
Brand
StemSpan
 
Area of Interest
Cancer, Immunology, Stem Cell Biology
 
Formulation Category
Serum-Free
 

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Data Figures

StemSpan™ NK procedure from seeding CB-derived CD34+ cells to harvesting of differentiated NK cells

Figure 1. StemSpan™ NK Cell Generation Protocol

CB-derived CD34+ cells are seeded on day 0. Medium should be topped up after 3 - 4 days of culture followed by two half-medium changes every 3 - 4 days. On day 14, cells at the lymphoid progenitor stage are harvested and reseeded for further differentiation into NK cells. Top-up and half-medium changes should be performed every 3 - 4 days after harvest and reseed, as indicated in the figure. Note: UM729 should only be added to the NK Cell Differentiation Medium, but not the Lymphoid Progenitor Expansion Medium.

Frequency and yield of CD56- and NKp46-expressing cells produced after culture of CD34+ HSPCs with the StemSpan™ NK Cell Generation Kit

Figure 2. Frequency and Yield of CD56+ NK Cells After 28 Days of Culture

CB-derived CD34+ cells (freshly isolated or frozen) were cultured with the StemSpan™ NK Cell Generation Kit for 28 days as described. Cells were harvested and analyzed for (A,B) CD56 and (A) NKp46 expression by flow cytometry. Dead cells were excluded by light scatter profile and viability staining. (B) The average frequency of viable CD56+ NK cells on day 28 was 77%, with ~9,000 CD56+ cells produced per input CB-derived CD34+ cell. Shown are means with 95% confidence intervals (n = 45: 23 freshly isolated and 22 frozen CD34+ cell samples). BM-derived CD34+ cells were also differentiated into NK cells using the StemSpan™ NK Cell Generation Kit. The yield of NK cells from BM HSPCs is typically lower than with CB, averaging ~75 per input CD34+ cell (n = 3, data not shown).

Flow cytometry plots showing cell surface marker expression on CD56+ NK cells after culture with the StemSpan™ NK Cell Generation Kit

Figure 3. Cell Surface Marker Expression on CD56+ NK Cells After 28 Days of Culture

CB-derived CD34+ cells were cultured with the StemSpan™ NK Cell Generation Kit for 28 days. The differentiated cells were harvested and analysed by flow cytometry for the expression of CD56, NKp44, NKp30, NKG2D, CD94, CD16, and KIR. Staining for KIR molecules was performed using a combination of two clones for the antibody, 180704 and HP-MA4, as each recognizes a distinct subset of KIR molecules.

Killing activity of NK cells toward K562 target cells compared to PB NK cells following culture with the StemSpan™ NK Cell Generation Kit

Figure 4. Cultured NK Cells Exhibit Cytotoxicity Toward K562 Cell Line

NK cells were generated from CB-derived CD34+ cells over 28 days using the protocol in Figure 1. On day 28, cells were harvested, stained for CD56, and viable CD56+ cells were counted. K562 cells were incubated with 8 μM calcein AM at 37°C for 1 hour and then washed twice. CD56+ NK cells were then combined with 10,000 of these calcein AM-labeled K562 target cells at an Effector:Target ratio of 5:1 in U-bottom 96-well plates and co-cultured at 37°C for 4 hours. Adult peripheral blood (PB) NK cells and monocytes isolated using EasySep™ were used as positive and negative controls, respectively. PB NK cells were cultured overnight with the NK Cell Differentiation Supplement and SFEM II, while PB monocytes were cultured overnight in SFEM II only. To detect spontaneous release, control wells containing only calcein AM-labeled K562 target cells were set up. The labeled K562 cells were treated with 1% Triton™ X-100 to measure maximum release. After incubation, plates were centrifuged at 500 x g for 5 minutes and 100 μL of supernatant was transferred to black plates and analyzed using a SpectraMax® microplate reader (excitation 485 nm/emission 530 nm). Results are expressed as % specific lysis: [(test release - spontaneous release) x 100] / (maximum release - spontaneous release). CB CD34+-derived NK cells show similar killing activity toward K562 target cells compared to PB NK cells. Shown are means ± SD (CB CD34+-derived NK cells: n = 18, PB NK cells and monocytes: n = 7).

Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

Document Type
Product Name
Catalog #
Lot #
Language
Document Type
Product Information Sheet
Product Name
StemSpan™ NK Cell Differentiation Supplement (100X)
Catalog #
09950
Lot #
All
Language
English
Document Type
Technical Manual
Product Name
StemSpan™ NK Cell Differentiation Supplement (100X)
Catalog #
09950
Lot #
All
Language
English
Document Type
Safety Data Sheet
Product Name
StemSpan™ NK Cell Differentiation Supplement (100X)
Catalog #
09950
Lot #
All
Language
English

Applications

This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

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Workflow Stages

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Species Human
Formulation Category Serum-Free
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