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Heat-Stable FGFs for Improved Cell Culture Potency
Engineered for greater stability & efficiency
Heat-stable recombinant proteins have been engineered for greater stability in cell culture, exhibiting enhanced bioactivity at 37°C. Because Heat Stable proteins are bioactive longer than native proteins in standard cell culture conditions, researchers may be able to use less to maintain the same growth rates, therefore improving the efficiency of their cell culture applications.
- Superior performance—retains bioactivity without the use of artificially high concentrations
- Improved efficiency—the stable bioactivity means you won’t have to supplement with additional protein (a necessary step when using native proteins), and you’ll save money in reagent costs over the longer term
- Easy to use—direct replacement for your current proteins allowing for easy substitution in protocols
- Heat Stable Proteins available—Heat Stable bFGF and Heat Stable FGF-10.
Superior cell culture performance
Heat Stable FGFs maintain activity at 37°C
Figure 1. Cell proliferation studies demonstrate Heat Stable FGFs exhibit superior activity compared to native FGFs. HS bFGF activity was measured with PrestoBlue dye analysis of Balb/3T3 cell proliferation (top panels). The 10 ng/mL HS bFGF (top left) and native bFGF (top right) solutions were stored at 4°C (unstressed) or 37°C (heat stressed) for 72 hours before being used to treat the Balb/3T3 cells in a dose-dependent manner. Likewise, HS FGF10 activity was measured with PrestoBlue HS Cell Viability reagent analysis of MCF7 cell proliferation (bottom panels). The 1 μg/mL HS FGF10 (bottom left) and native FGF10 (bottom right) solutions were stored at 4°C (unstressed) or 37°C (heat stressed) for 72 hours before being used to treat the MCF7 cells in a dose-dependent manner.
Over 80% sustained biological activity
Figure 2. Heat Stable FGF growth factors demonstrate greater activity after 72 hours of heat stress than native proteins. (Left) HS bFGF activity was measured with PrestoBlue dye analysis of Balb/3T3 cell proliferation. (Right) HS FGF10 activity was measured with PrestoBlue HS Cell Viability reagent analysis of MCF7 cell proliferation. The percent activity is the amount of activity maintained after heat stress relative to the same solution stored at 4°C (unstressed). The 10 ng/mL HS bFGF and native bFGF, and 1 μg/mL HS FGF10 and native FGF10 solutions were stored at 4°C or 37°C for 72 hours before being used to treat Balb/3T3 or MCF7 cells, respectively.
Figure 3. Cell proliferation assay demonstrating how biological activity of tag-free heat stable PeproTech bFGF protein is maintained longer under cell culture conditions compared to the equivalent native bFGF protein.
HS bFGF offers increased efficiency
Figure 4. Heat Stable FGFs allows normal proliferation of cells with growth factor. (A) 5 ng/mL of bFGF leads to significantly slower neural stem cell (NSC) proliferations as compared with 5 ng/mL HS bFGF, which maintains a proliferation rate equivalent to that of 20 ng/mL native bFGF. Mean +/- SEM. ** = p < 0.01. (B) Additionally, while 5 ng/mL native bFGF leads to neurite outgrowth (denoted by red arrows), HS bFGF maintains multipotent NSC morphology. (C) Relative activity of FGF10 was measured following incubation of 1 μg/mL HS FGF10 and native FGF10 stock solutions at 4°C or 37°C for 72 hours before being used to treat MCF7 cells. Activity measured with PrestoBlue HS Cell Viability reagent demonstrated 10 ng/mL of HS FGF10 performed equivalently to 100 ng/mL of native FGF10. Mean +/- SEM. *** = p < 0.0001; n.s. = not significant.
HS bFGF maintains neural stem cell multipotency
Figure 5. Heat Stable bFGF maintains the morphology and SOX1 expression of multipotent NSCs. Phase contrast (left) and immunostained (right) photomicrographs of NSCs cultured in the presence of HS bFGF (top row) and native bFGF (bottom row). Immunostaining for NSC multipotency marker SOX1 (red) with nuclear (DAPI) counterstain (blue).
HS bFGF does not interfere with undirected differentiation
Figure 6. Spontaneous differentiation supported by HS bFGF. Primary rat NSC isolated from Sprague Dawley E14 cortex cultured for 3 passages in DMEM/F12 + GlutaMAX supplemented with N2, NEAA, β-mercaptoethanol and 10 ng/mL HS bFGF. Media changes made every 48 hours without daily spiking of bFGF. At passage 3, cells were allowed to spontaneously differentiate by culturing in growth medium without HS bFGF. Cells showed equivalent potential to undergo undirected differentiation as compared to native bFGF, measured by markers MAP2 (neuron), GFAP (astrocytes) and GALC (oligodendrocytes).
HS bFGF enhances growth of cancer spheroids via a more streamlined protocol
Figure 7. HS bFGF enhances growth of cancer spheroids without the need for troublesome media changes. Spheroids were formed in Nunclon Sphera microplates in serum-free medium containing no bFGF, 10 ng/mL native bFGF, or 10 ng/mL HS bFGF. After 8 days in culture without media changes, the cells were stained via live (green)-dead (red) assay and DAPI counterstain (blue). All spheroids were viable, but the spheroid exposed to HS bFGF was the largest of the three experimental conditions. For more information, please see our application note
Gene expression of cells treated with HS bFGF is comparable to that of cells treated with native bFGF
Click image to enlarge
Figure 8. bFGF-target and off-target gene expression was comparable in cells treated with native bFGF and Heat Stable bFGF. The TaqMan Array Human Signal Transduction Pathways array (Cat. No. 4418775) and the TaqMan Array Human FGF Pathway (Cat. No. 4414136) were used to assess expression of genes in the androgen, calcium, CREB, estrogen, hedgehog, insulin, JAK-STAT, LDL, mitogenic, NFAT, NFkB, p53, phospholipase C, protein kinase C, retinoic acid, stress, survival, TGF-β, Wnt, and FGF-associated pathways, respectively. Expression was assessed using human induced pluripotent stem cells treated with 10 ng/mL native bFGF or Heat Stable bFGF. Genes from both arrays were separated into ‘bFGF-target’ or ‘off-target’ based on their downstream relationship to bFGF, as is currently indicated by the literature. The bFGF-target and off-target panels show the results from 43 and 71 genes, respectively.
HS bFGF outperforms top competitors
Figure 9. HS bFGF exhibits superior activity after extended 37°C incubation compared to competitor’s products. Activity was measured with PrestoBlue dye analysis of Balb/3T3 cell proliferation. The percent activity is amount of activity maintained after heat stress (37°C), relative to the same solution stored at 4°C (unstressed). The 10 ng/mL HS bFGF and competitor bFGF solutions were stored at 4°C or 37°C for 72 hours before being used to treat the Balb/3T3 cells, N≥3. Mean ± SEM.
HS bFGF facilitates stem cell culture workflows
Figure 10. Comparison iPSCs cultured in the presence of Gibco HS bFGF or StemBeads reagent. Microscopic images of StemBeads reagent (10 ng/mL) captured at (A) 10x and (B) 20x magnification. Because StemBeads beads vary in size (arrows indicate different sized StemBeads particles), they can be difficult to distinguish from cells and also from debris. Twenty-four hours after seeding human iPSCs in the presence of either (C) 10 ng/mL Gibco HS bFGF or (D) 10 ng/mL StemBeads reagent, phase-contrast images were captured of the cultured cells. The image of cells cultured with HS bFGF is clear, containing minimal debris, whereas the StemBeads reagent–containing culture shows more apparent debris. In addition because the beads tend to sink relatively quickly in solution, accurately measuring StemBeads reagent concentration can present a challenge.
Heat Stable FGFs maintain activity at 37°C
Figure 1. Cell proliferation studies demonstrate Heat Stable FGFs exhibit superior activity compared to native FGFs. HS bFGF activity was measured with PrestoBlue dye analysis of Balb/3T3 cell proliferation (top panels). The 10 ng/mL HS bFGF (top left) and native bFGF (top right) solutions were stored at 4°C (unstressed) or 37°C (heat stressed) for 72 hours before being used to treat the Balb/3T3 cells in a dose-dependent manner. Likewise, HS FGF10 activity was measured with PrestoBlue HS Cell Viability reagent analysis of MCF7 cell proliferation (bottom panels). The 1 μg/mL HS FGF10 (bottom left) and native FGF10 (bottom right) solutions were stored at 4°C (unstressed) or 37°C (heat stressed) for 72 hours before being used to treat the MCF7 cells in a dose-dependent manner.
Over 80% sustained biological activity
Figure 2. Heat Stable FGF growth factors demonstrate greater activity after 72 hours of heat stress than native proteins. (Left) HS bFGF activity was measured with PrestoBlue dye analysis of Balb/3T3 cell proliferation. (Right) HS FGF10 activity was measured with PrestoBlue HS Cell Viability reagent analysis of MCF7 cell proliferation. The percent activity is the amount of activity maintained after heat stress relative to the same solution stored at 4°C (unstressed). The 10 ng/mL HS bFGF and native bFGF, and 1 μg/mL HS FGF10 and native FGF10 solutions were stored at 4°C or 37°C for 72 hours before being used to treat Balb/3T3 or MCF7 cells, respectively.
Figure 3. Cell proliferation assay demonstrating how biological activity of tag-free heat stable PeproTech bFGF protein is maintained longer under cell culture conditions compared to the equivalent native bFGF protein.
HS bFGF offers increased efficiency
Figure 4. Heat Stable FGFs allows normal proliferation of cells with growth factor. (A) 5 ng/mL of bFGF leads to significantly slower neural stem cell (NSC) proliferations as compared with 5 ng/mL HS bFGF, which maintains a proliferation rate equivalent to that of 20 ng/mL native bFGF. Mean +/- SEM. ** = p < 0.01. (B) Additionally, while 5 ng/mL native bFGF leads to neurite outgrowth (denoted by red arrows), HS bFGF maintains multipotent NSC morphology. (C) Relative activity of FGF10 was measured following incubation of 1 μg/mL HS FGF10 and native FGF10 stock solutions at 4°C or 37°C for 72 hours before being used to treat MCF7 cells. Activity measured with PrestoBlue HS Cell Viability reagent demonstrated 10 ng/mL of HS FGF10 performed equivalently to 100 ng/mL of native FGF10. Mean +/- SEM. *** = p < 0.0001; n.s. = not significant.
HS bFGF maintains neural stem cell multipotency
Figure 5. Heat Stable bFGF maintains the morphology and SOX1 expression of multipotent NSCs. Phase contrast (left) and immunostained (right) photomicrographs of NSCs cultured in the presence of HS bFGF (top row) and native bFGF (bottom row). Immunostaining for NSC multipotency marker SOX1 (red) with nuclear (DAPI) counterstain (blue).
HS bFGF does not interfere with undirected differentiation
Figure 6. Spontaneous differentiation supported by HS bFGF. Primary rat NSC isolated from Sprague Dawley E14 cortex cultured for 3 passages in DMEM/F12 + GlutaMAX supplemented with N2, NEAA, β-mercaptoethanol and 10 ng/mL HS bFGF. Media changes made every 48 hours without daily spiking of bFGF. At passage 3, cells were allowed to spontaneously differentiate by culturing in growth medium without HS bFGF. Cells showed equivalent potential to undergo undirected differentiation as compared to native bFGF, measured by markers MAP2 (neuron), GFAP (astrocytes) and GALC (oligodendrocytes).
HS bFGF enhances growth of cancer spheroids via a more streamlined protocol
Figure 7. HS bFGF enhances growth of cancer spheroids without the need for troublesome media changes. Spheroids were formed in Nunclon Sphera microplates in serum-free medium containing no bFGF, 10 ng/mL native bFGF, or 10 ng/mL HS bFGF. After 8 days in culture without media changes, the cells were stained via live (green)-dead (red) assay and DAPI counterstain (blue). All spheroids were viable, but the spheroid exposed to HS bFGF was the largest of the three experimental conditions. For more information, please see our application note
Gene expression of cells treated with HS bFGF is comparable to that of cells treated with native bFGF
Click image to enlarge
Figure 8. bFGF-target and off-target gene expression was comparable in cells treated with native bFGF and Heat Stable bFGF. The TaqMan Array Human Signal Transduction Pathways array (Cat. No. 4418775) and the TaqMan Array Human FGF Pathway (Cat. No. 4414136) were used to assess expression of genes in the androgen, calcium, CREB, estrogen, hedgehog, insulin, JAK-STAT, LDL, mitogenic, NFAT, NFkB, p53, phospholipase C, protein kinase C, retinoic acid, stress, survival, TGF-β, Wnt, and FGF-associated pathways, respectively. Expression was assessed using human induced pluripotent stem cells treated with 10 ng/mL native bFGF or Heat Stable bFGF. Genes from both arrays were separated into ‘bFGF-target’ or ‘off-target’ based on their downstream relationship to bFGF, as is currently indicated by the literature. The bFGF-target and off-target panels show the results from 43 and 71 genes, respectively.
HS bFGF outperforms top competitors
Figure 9. HS bFGF exhibits superior activity after extended 37°C incubation compared to competitor’s products. Activity was measured with PrestoBlue dye analysis of Balb/3T3 cell proliferation. The percent activity is amount of activity maintained after heat stress (37°C), relative to the same solution stored at 4°C (unstressed). The 10 ng/mL HS bFGF and competitor bFGF solutions were stored at 4°C or 37°C for 72 hours before being used to treat the Balb/3T3 cells, N≥3. Mean ± SEM.
HS bFGF facilitates stem cell culture workflows
Figure 10. Comparison iPSCs cultured in the presence of Gibco HS bFGF or StemBeads reagent. Microscopic images of StemBeads reagent (10 ng/mL) captured at (A) 10x and (B) 20x magnification. Because StemBeads beads vary in size (arrows indicate different sized StemBeads particles), they can be difficult to distinguish from cells and also from debris. Twenty-four hours after seeding human iPSCs in the presence of either (C) 10 ng/mL Gibco HS bFGF or (D) 10 ng/mL StemBeads reagent, phase-contrast images were captured of the cultured cells. The image of cells cultured with HS bFGF is clear, containing minimal debris, whereas the StemBeads reagent–containing culture shows more apparent debris. In addition because the beads tend to sink relatively quickly in solution, accurately measuring StemBeads reagent concentration can present a challenge.
Ordering information
FAQs
| Question | Answer |
|---|---|
| What is the difference between the Heat Stable bFGF products? | A His tag is present on products with catalog numbers of PHG0367, PHG0368, PHG0369, and PHG0360. No His tag is present on products with catalog numbers of 100-18BHS-100UG, 100-18BHS-10UG, 100-18BHS-1MG, 100-18BHS-250UG, 100-18BHS-500UG, and 100-18BHS-50UG. |
| Is Heat Stable Recombinant Human bFGF full length? | Yes, Heat Stable Recombinant Human bFGF is full-length at 155 amino acids, plus 20 amino acids for N-terminal tag for the his-tagged versions of the protein. See catalog numbers in the FAQ above. |
| Can Heat Stable Recombinant Human bFGF be used in place of native recombinant human bFGF? | Yes, Heat Stable Recombinant Human bFGF can be used in place of native human bFGF. It is a direct replacement, offering greater stability in cell culture conditions. |
| Has protein engineering altered the activity of Heat Stable Recombinant Human bFGF? | HS bFGF does not exhibit higher bioactivity, nor does it stimulate cell signaling differently than native bFGF. |
| Why should Heat Stable Recombinant Human bFGF be used instead of native recombinant human bFGF? | Native human bFGF degrades at standard culture conditions (i.e., 37°C). This means that more frequent media changes and/or more protein is required to maintain biological activity. Heat Stable Recombinant Human bFGF retains bioactivity minimizing fluctuations to more closely mimic physiological conditions for cells. |
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