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Detection reagent for undifferentiated human ES cells and human iPS cells 
If only it is added to the culture fluid, human ES cells and human iPS cells can be detected!
Fluorescent-labeled rBC2LCN(AiLecS1)

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rBC2LCN (AiLecS1) is a recombinant lectin expressing the N-terminal domain of BC2L-C, which is a lectin derived from Burkholderia cenocepacia, in Escherichia coli. rBC2LCN has a high specificity to sugar chains present on the cell surface of undifferentiated human ES cells and human iPS cells.

This product has been labeled with a fluorochrome, and by being added to the culture fluid of human ES cells or human iPS cells it is possible to analyze undifferentiated cells alive, therefore useful as markers for detecting undifferentiated cells. It can also be used for both cell staining and flow cytometry. In addition, no cytotoxicity has been detected in continuous culture for several days in the presence of this product.

We offer the line-up of three types of Fluorescein Isothiocyanate Isomer (FITC), Yellow fluorescent label, (Cy3 area), Red fluorescent label (Cy5 area).


1. Link

2. Features

  • Capable of staining by only added to a culture medium
  • Capable of staining live cells clearly without cell fixation
  • Has a low cytotoxicity and can be cultured in stained condition
  • Recognizes the sugar chain on the surface of cells in the same manner as Tra-1-60, Tra-1-81, SSEA-3 and SSEA-4
  • Usable for cell staining and flow cytometry

3. Product Outline

  • The sterility test has done. (filtered and sterilized by 0.1 μm filter)
  • PBS (-) solution
  • Practical dilution ratio
    Live Cell Imaging 1:100 ~ 1,000
    Flow Cytometry 1:100 ~ 1,000
  • Excitation wavelength, emission wavelength

  • Excitation Emission
    rBC2LCN-FITC 495 nm 520 nm
    rBC2LCN-547 551 nm 565 nm
    rBC2LCN-635 634 nm 654 nm

4. How to use

4-1. Cell staining

 ■ Live cell staining (Live Cell Imaging)

  1. Prepare the human ES cells and human iPS cells during culture.
  2. Add 1 ~ 10 μL of Fluorescent labeling rBC2LCN per 1 mL of culture medium.
  3. Incubate them for 30 minutes at 37°C under CO2 of 5 % concentration.
  4. Replace them with the new medium or HBSS (+).
  5. Observe them using a fluorescence microscope.
  ■ Staining of fixed cells
  1. Prepare the human ES cells and human iPS cells during culture.
  2. Remove the medium and wash them with HBSS (+).
  3. Add paraformaldehyde solution (PFA) of 4 % concentration to them and leave them statically for 10-20 minutes at room temperature.
  4. After removal of PFA, wash them three times with D-PBS (-).
  5. Add D-PBS (-) to them.
  6. Add 1 ~ 10μL of Fluorescent labeling rBC2LCN per 1mL of D-PBS (-).
  7. Incubate them for 30 minutes at 37°C under CO2 of 5 % concentration.
  8. Replace them with D-PBS (-) .
  9. Observe them using a fluorescence microscope.

4-2. Flow cytometry

  1. Prepare the human ES cells and human iPS cells during culture.
  2. Disperse them to a single cell using the cell dispersion solution.
  3. Transfer the cell dispersion solution to a tube and centrifuge it for 3 minutes at 1,000rpm.
  4. After removing the supernatant, suspend the cells in the FCM Buffer* and discard the supernatant after centrifugation.
    * FCM Buffer: D-PBS (-), HBSS (-) or those containing 10mmol/ L EDTA and 1% BSA.
  5. Suspend them in the FCM Buffer so as to be 5 X 106 cells/mL.
  6. Add 1 ~ 10μL of Fluorescent label rBC2LCN per 1mL of cell suspension.
  7. Shield the light and leave them statically for 30 minutes at room temperature.
  8. Centrifuge them for 3 minutes at 1,000rpm and discard the supernatant.
  9. Suspend the cells in the FCM Buffer and discard the supernatant after centrifugation.
  10. Resuspend them in an appropriate amount of FCM Buffer.
  11. Subject them to flow cytometry.
  [Precautions for use]
  1. Staining with rBC2LCN will be maintained for 2 or 3 days.
  2. When you use a culture medium containing serum, there is a possibility that the background of the signal becomes higher.
  3. In case of doing the sorting over cells by flow cytometry, please add Y-27632 to them so as to be at 10 μmol/L final concentration at the time of cell dispersion or FCM Buffer.

5. Live cell staining of human iPS cells (Live Cell Imaging)

■ Human iPS 201B7 strain cells were stained with rBC2LCN-FITC, rBC2LCN-635 and Hoechst 33342 (without cell fixation).

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                                          (Dilution ratio 1: 1,000)

■ Human iPS 201B7 strain cells were stained with rBC2LCN-FITC, Tra-1-60, Tra-1-81 and SSEA-4, then the stained image was confirmed after 2 hours (without cell fixation).

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(Dilution ratio 1: 1,00)

 

★ rBC2LCN has a stronger intensity of live cell staining compared with Tra-1-60 and Tra-1-81.

★The staining with rBC2LCN will be sustained vividly up to the second day even after exchange of the culture medium.


6. Live cell staining of human ES cells (Live Cell Imaging)

Human ES WA01 strain cells were stained with rBC2LCN-FITC (without cell fixation). A portion of the cells differentiates without being able to maintain the undifferentiated state. In the case of live cell staining with rBC2LCN-FITC, it is confirmed that the portion maintaining the undifferentiated state is stained though, the differentiated portion is unstained.

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< Data providers: The National Institute of Advanced Industrial Science and Technology Biotechnology Research Institute for Drug Discovery Stem Cell Engineering Research Group Yasuko Konuma, Yuzuru Ito >

7. Staining fixed human iPS cells

After fixation of human iPS 201B7 strain cells by paraformaldehyde, cell staining was done with rBC2LCN and DAPI.

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8. Cytotoxicity evaluation of human iPS cells

The culture was continued in the state of adding rBC2LCN-FITC of 1 / 1,000,1 / 100 or 1 / 50 amount of the culture fluid to that of human iPS 201B7 strain cells. As a result, regardless of the concentration of rBC2LCN-FITC, it was confirmed to exhibit a comparable cell proliferation at each concentration.

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[Cell strain]
Human iPS 201B7 strain cells

[Culture medium composition]
StemSure hPSC medium Δ + 35ng / mL bFGF

[Coating]
Matrigel® hESC-Qualified Matrix

[The number of cell seeding]
4 x 104cells/well (using 12-well plate)

9. Separation of human iPS cells using Flow Cytometry

Human iPS 201B7 strain cells and human normal diploid fibroblasts were stained with rBC2LCN-FITC and rBC2LCN-635, then were subjected to flow cytometry. As a result, it was possible to separate undifferentiated human iPS cells from differentiated human diploid fibroblasts.

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10. References

  • Onuma, Y., et al.: Biochem. Biophys. Res. Commun., 431, 524, (2013).
  • Tateno, H., et al.: Stem Cells Transl. Med., 2, 265, (2013).
  • Tateno, H., et al.: Sci. Rep., 4, 4069, (2014).

11. Product List

Product Name Package Size Wako Catalog No. Grade Storage Condition
rBC2LCN-FITC 【AiLecS1-FITC】
  Ex. 495 nm, Em. 520 nm
100 μL
100 μL x 5
180-02991
186-02993
for Cell Staining Keep at -20°C.
rBC2LCN-547 【AiLecS1-547】
  Ex. 551 nm, Em. 565 nm
100 μL
100 μL x 5
186-03211
182-03213
rBC2LCN-635 【AiLecS1-635】
  Ex. 634 nm, Em. 654 nm
100 μL
100 μL x 5
185-03161
181-03163
 

12. Related Products

12-1. rBC2LCN stripping solution

This product can peel off the rBC2LCN lectin bound to sugar chains present on the cell surface of the undifferentiated cells from the cell. After peeling off rBC2LCN, it is possible to stain the cells with other antibody and continue the culture.

Product Name Package Size Wako Catalog No. Grade Storage Condition
rBC2LCN Stripping Solution 【AiWashS1】
10 mL 182-03171
for Cell Culture Keep at 2-10°C.


12-2. BC2LCN lectin, recombinant, solution

We also offer the line-up of unlabeled rBC2LCN.

Product Name Package Size Wako Catalog No. Grade Storage Condition
BC2LCN 【AiLecS1】 Lectin, recombinant, Solution
1 mg
1 mg x 5
029-18061
025-18063
for Glycobiology Keep at -20°C.


12-3. StemSure hPSC medium Δ

This is a serum-free liquid culture medium which can be used for maintainance and culture of human pluripotent stem cells (human ES cells and human iPS cells) under the feeder-free conditions. The ingredients including the materials derived from animal are not used as a raw material.

Product Name Package Size Wako Catalog No. Grade Storage Condition
StemSure hPSC Medium Δ
100 mL
100 mL x 4
197-17571
193-17573
for Cell Culture Keep at -20°C.
Fibloblast Growth Factor (basic), Human, recpmbonant, Animal-derived-free 【bFGF/FGF2】
50 μg
100 μg
1 mg
064-05381
068-05384
060-05383
for Cellbiology
hPSC Dissociation Solution
100 mL 160-27051 for Cell Culture Keep at 2-10°C.


12-4. StemSure series

The quality test is performed by using mouse ES D3 strain cells .

Product Name Pkg. Size Wako Cat. No. Grade Storage Condition
StemSure D-MEM with Phenol Red and Sodium Pyruvate
500 mL 197-16275 for Cell Culture Keep at 2-10°C.
StemSure Serum Replacement
500 mL 197-16775 Keep at -20°C.
StemSure 10 mmol/L 2-Mercaptoethanol Solution (x 100)
100 mL 198-15781
StemSure 50 mmol/L Monothioglycerol Solution (x 100)
A reducing agent that can be used in equivalent to 2ME.
Not classified as a poison.
100 mL 195-15791
StemSure 0.1 w/v% Gelatin Solution
500 mL 190-15805 Keep at room temperature.
StemSure Freezing Medium
A cryopreservation solution for ES cells and iPS cells containing the BSA.
100 mL 195-16031 Keep at 2-10°C.
StemSure hPSC Freezing Medium, AF
An animal-free cryopreservation solution for ES cells and iPS cells.
100 mL 197-17831
StemSure LIF, Mouse, recombinant, Solution
106 units
106 units x 10
199-16051
195-16053
Keep at -20°C.


12-5. Reagents for ES and iPS cells research   [Brochure ]

After the announcement of the establishment of iPS cells in 2007, large numbers of literature related to iPS cells have been published. We offer the line-up of the low-molecule compounds which have been reported in a variety of literature to be involved in maintenance of undifferentiated potency and induction of differentiation of ES cells and iPS cells. [Link]

12-6. Reagents for liquid culture medium and cell culture

We offer an abundant line-up of products such as the liquid culture medium, the balanced salt solution, the solution for peeling and dispersion of cells, antibiotic solution and extracellular matrix etc.. [Link]

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