Fluorescent-labeled rBC2LCN (Animal-derived-free)

• Animal-derived-free : manufactured without animal-derived raw materials
• Enables cell staining simply by adding to the culture medium
• Allows clear staining of live cells without fixation
• Suitable for both cell staining and flow cytometry
• Low cytotoxicity, allowing continued culture after staining
• Recognizes cell-surface glycans, similar to Tra-1-60 and SSEA-4

• Sterility tested
• Supplied in PBS(-) solution
• Recommended dilution
  Live-cell staining: 1:100 – 1:1,000
  Flow cytometry: 1:100 – 1:1,000
• Excitation / Emission wavelengths:

Staining Live Cells (Live-cell Imaging)

1. Prepare human ES/iPS cells in culture.
2. Add 1–10 μL of rBC2LCN-FITC, AF or rBC2LCN-635, AF per 1 mL of culture medium.
3. 37 ℃, 5 % CO₂for 30 minutes.
4. Replace the medium with fresh medium, HBSS(+), or D-PBS(-).
5. Observe using a fluorescence microscope.

Staining Fixed Cells

1. Prepare human ES/iPS cells in culture.
2. Remove the culture medium and wash cells with HBSS(+).
3. Add 4 % paraformaldehyde (PFA) solution and incubate at room temperature for 10–20 minutes.
4. Remove PFA and wash three times with D-PBS(-).
5. Add D-PBS(-).
6. Add 1–10 μL of rBC2LCN-FITC, AF or rBC2LCN-635, AF per 1 mL of D-PBS(-) .
7. Incubate at 37 ℃, 5 % CO_₂ for 30 minutes.
8. Replace with D-PBS(-).
9. Observe using a fluorescence microscope.

Flow Cytometry

1. Prepare human ES/iPS cells in culture.
2. Dissociate cells into single-cell suspension using a cell dissociation reagent.
3. Transfer the cell suspension to a tube and centrifuge at 1,000 rpm for 3 minutes.
4. Discard the supernatant, resuspend cells in FCM Buffer^*, centrifuge again, and discard the supernatant.
   ^*FCM Buffer: D-PBS(-) or HBSS(-), optionally supplemented with 10 mmol/L EDTA and 1 % BSA or FBS.
5. Resuspend cells in FCM Buffer at 5x10^⁶ cells/mL.
6. Add 1–10 μL of fluorescently labeled rBC2LCN per 1 mL of cell suspension.
7. Incubate for 30 minutes at room temperature in the dark.
8. Centrifuge at 1,000 rpm for 3 minutes and discard the supernatant.
9. Resuspend cells in FCM Buffer, centrifuge again, and discard the supernatant.
10. Resuspend cells in an appropriate volume of FCM Buffer.
11. Analyze by flow cytometry.

【Precautions for Use】

• Staining with rBC2LCN can be maintained for 2–3 days.
• When using serum-containing media, background fluorescence may increase.
• When sorting cells by flow cytometry, add Y-27632 during cell dissociation and to the FCM buffer at a final concentration of 10 μmol/L.

Human iPS cell line 201B7 was stained without fixation using rBC2LCN, SSEA-4, and Tra-1-60. Images were acquired 2 hours after staining. Compared with staining using the other antibodies, rBC2LCN enabled clearer visualization of the cells.

Human iPS cell line 201B7 was fixed with paraformaldehyde and stained with rBC2LCN, Oct3/4, and DAPI. Compared with live-cell staining, the cells were stained more clearly.

Human iPS cell line 201B7 was cultured with rBC2LCN-FITC, AF at 1/1,000, 1/100, and 1/50 of the culture medium volume. Cell proliferation was comparable to the untreated control at all concentrations of rBC2LCN-FITC, AF. In addition, cell morphology remained similar between treated and untreated cells.

Human iPS cell line 201B7 and normal diploid human fetal lung fibroblasts were stained with rBC2LCN-FITC, AF or rBC2LCN-635, AF and analyzed by flow cytometry. The results show that undifferentiated human iPS cells can be distinguished from differentiated human diploid fibroblasts.

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