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StemSpan™ B细胞分化添加物4（20X）

添加物3（20X）与添加物4（20X）搭配使用，以将 B 细胞祖细胞分化为 CD19+IgM+ B 细胞、记忆样 B 细胞和抗体分泌细胞
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产品号 #（选择产品）

产品号 #100-1253_C

添加物3（20X）与添加物4（20X）搭配使用，以将 B 细胞祖细胞分化为 CD19+IgM+ B 细胞、记忆样 B 细胞和抗体分泌细胞

产品优势

  • 高效高产量获取 CD19+IgM+ B 细胞、记忆样 B 细胞和抗体分泌细胞；
  • 作为 StemSpan™ B 细胞生成试剂盒方案的一部分使用
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总览

StemSpan™ B 细胞分化添加物3（20X）支持CD19+ B细胞祖细胞的分化。将此添加物与StemSpan™ B细胞分化添加物4（20X）搭配使用，可选择性促进B细胞祖细胞分化为CD19+IgM+ B细胞、记忆样B细胞和抗体分泌细胞（ASC）。

此添加物可单独购买，也可作为StemSpan™ B细胞生成试剂盒的一部分购买，并可与StemSpan™无血清扩增培养基 II 搭配使用，以选择性地将B细胞祖细胞分化为B细胞和 ASC。

亚型
添加剂
 
细胞类型
B 细胞，造血干/祖细胞
 
种属
人
 
应用
细胞培养，分化
 
品牌
StemSpan
 
研究领域
免疫
 
制剂类别
无血清
 

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Data Figures

StemSpan™ B Cell Generation Protocol

Figure 1. StemSpan™ B Cell Generation Protocol

Cord blood-derived CD34+ HSPCs are seeded on Day 0 in StemSpan™ SFEM II (Catalog #09605) containing StemSpan™ B Cell Differentiation Supplement 1 (Catalog #100-1251) and cultured for 14 days, followed by StemSpan™ SFEM II containing StemSpan™ B Cell Differentiation Supplement 2 (Catalog #100-1252) for an additional 14 days. Medium should be topped up after 3 - 4 days of culture followed by two half-medium changes every 3 - 4 days. On Day 28, cells are harvested and reseeded in StemSpan™ SFEM II containing StemSpan™ B Cell Differentiation Supplement 3 (Catalog #100-1253), followed by a top up with StemSpan™ SFEM II containing StemSpan™ B Cell Differentiation Supplement 4 (Catalog #100-1254) after 3 days for further differentiation to B cells.

Flow Cytometry of CD34+ cells (freshly isolated or from cryopreserved stock) were cultured in StemSpan™ SFEM II containing StemSpan™ B Cell Differentiation Supplement 1 for 14 days.

Figure 2. B Lymphoid Progenitors Are Contained Within the Lineage Negative Cell Population After 14 Days of Culture

Cord blood-derived CD34+ cells (freshly isolated or from cryopreserved stock) were cultured in StemSpan™ SFEM II (Catalog # 09605) containing StemSpan™ B Cell Differentiation Supplement 1 (Catalog #100-1251) for 14 days. (A-D) Cells were harvested and analyzed by flow cytometry for the presence of lineage-negative cells (Lin-, to exclude non-B lineage cells) and the expression of B lymphoid progenitor markers including CD34, CD10, CD19, and CD24. A small population of early B cell progenitors (which may contain proB and preB cells) can be detected on gated Lin- cells. (E) The average frequency of Lin- cells on Day 14 was 36.6 ± 1.4% with a yield of 47.1 ± 3.2 Lin- cells per input CD34+ cell. The graph shows mean with standard error (n = 36).

Flow cytometry and bar graphs showing the frequency and yield of CD19+ B cells after 28 days of culture

Figure 3. Frequency and Yield of CD19+ B Cells After 28 Days of Culture

Day 14 B lymphoid progenitors were further differentiated into CD19+ B cells by culturing for an additional 14 days in StemSpan™ SFEM II (Catalog #09605) containing StemSpan™ B Cell Differentiation Supplement 2 (Catalog #100-1252). (A-D) Cells were harvested and analyzed by flow cytometry for expression of CD10, CD19, CD20, CD24, and IgM within the Lin- cell population to detect later-stage B cell progenitors, containing PreB cells and a small population of IgM+ immature B cells. (E) The average frequency of CD19+ cells on Day 28 was 21.3 ± 1.6% with a yield of 82.7 ± 11.5 CD19+ cells per input CD34+ cell. The graph shows mean with standard error (n = 36)

Flow cytometry and bar graphs showing the frequency and yield of CD19+IgM+ and antibody-secreting B cells after 35 days of culture

Figure 4. Frequency and Yield of CD19+IgM+ and Antibody-Secreting B Cells After 35 Days of Culture

On Day 28, CD19+ B cells were further differentiated into CD19+IgM+ and antibody-secreting cells (ASCs) by culturing for an additional 7 days in StemSpan™ SFEM II (Catalog #09605) containing StemSpan™ B Cell Differentiation Supplement 3 (Catalog #100-1253) followed by StemSpan™ SFEM II containing StemSpan™ B Cell Differentiation Supplement 4 (Catalog #100-1254). (A-D) On Day 35, cells were analyzed by flow cytometry for the expression of CD19, CD20, CD24, CD27, CD38, and IgM. (E) The frequency and yield of CD19+ (pan B cells), CD19+IgM+ (IgM+ B cells), and CD19+CD27+CD38++ B cells (memory-like B cells and ASCs) are shown. On average, the frequencies and yield per input CD34+ cells were 54.0 ± 2.7%;140.7 ± 19.1, 27.3% ± 1.3%; 65.3 ± 9.9, and 14.2% ± 1.3%; 36.5 ± 5.3, respectively. The graph shows means with standard error (n = 36).

ELISpot assays and bar graphs displaying antibody-secreting B cells generated after 35 days of culture

Figure 5. Antibody-Secreting B Cells Generated After 35 Days of Culture

On Day 35, immunoglobulin-secreting cell frequencies of cultured B cells were determined by ELISpot assay. (A) Images of dual ELISpot assays (CTL ImmunoSpot®, IgM/IgG and IgA/IgE) for the detection of IgM (red) and IgG (blue) or IgA (red) and IgE (blue) antibody-secreting B cells (StemSpan™ SFEM II with StemSpan™ B Cell Differentiation Supplements 3 and 4, right) compared to negative control (StemSpan™ SFEM II without StemSpan™ B Cell Differentiation Supplements 3 and 4, left); 10,000 culture Day 35 cells per well were used. IgE ASCs were not detected. (B) The frequency and yield of ASCs are shown. On average 610 ± 67, 5 ± 1, and 317 ± 47 cells per 10,000 Day 35 cells secrete IgM, IgG, and IgA antibodies with a yield of 16.2 ± 2.4, 0.1 ± 0.04, and 5.2 ± 1.7 per input CD34+ cell, respectively. The graph shows mean with standard error (n = 8 - 33).

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™ B Cell Differentiation Supplement 3 (20X)
Catalog #
100-1253
Lot #
All
Language
English
Document Type
Safety Data Sheet
Product Name
StemSpan™ B Cell Differentiation Supplement 3 (20X)
Catalog #
100-1253
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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Resources and Publications

Educational Materials (2)

Human B Cell Isolation Product Selection
Brochure
Human B Cell Isolation Product Selection
Human B Cell Research Product Workflow
Brochure
Human B Cell Research Product Workflow

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Species Human
Formulation Category Serum-Free
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