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Culture and detection of primary cilia in endothelial cell models.

Lim YC, McGlashan SR, Cooling MT, Long DS - Cilia (2015)

Bottom Line: HMEC-1s also have shorter cilia compared to HUVECs (3.0 ± 1.0 μm versus 5.1 ± 2.4 μm, at confluence, p = 0.003).We recommend that future studies examining large blood vessel EC primary cilia use confluent HUVECs grown in high serum medium, as we found these cells to have a higher cilia incidence than low serum media HUVECs.For studies interested in microvasculature EC primary cilia, we recommend using cobblestone HMEC-1s grown in high serum medium, as these cells have a 19.5 ± 6.2 % cilia incidence.

View Article: PubMed Central - PubMed

Affiliation: Auckland Bioengineering Institute, University of Auckland, 70 Symonds Street, Auckland, 1142 New Zealand.

ABSTRACT

Background: The primary cilium is a sensor of blood-induced forces in endothelial cells (ECs). Studies that have examined EC primary cilia have reported a wide range of cilia incidence (percentage of ciliated cells). We hypothesise that this variation is due to the diversity in culture conditions in which the cells are grown. We studied two EC types: human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMEC-1s). Both cell types were grown in media containing foetal bovine serum (FBS) at high (20 % FBS and 10 % FBS for HUVECs and HMEC-1s, respectively) or low (2 % FBS) concentrations. Cells were then either fixed at confluence, serum-starved or grown post-confluence for 5 days in corresponding expansion media (cobblestone treatment). For each culture condition, we quantified cilia incidence and length.

Results: HUVEC ciliogenesis is dependent on serum concentration during the growth phase; low serum (2 % FBS) HUVECs were not ciliated, whereas high serum (20 % FBS) confluent HUVECs have a cilia incidence of 2.1 ± 2.2 % (median ± interquartile range). We report, for the first time, the presence of cilia in the HMEC-1 cell type. HMEC-1s have between 2.2 and 3.5 times greater cilia incidence than HUVECs (p < 0.001). HMEC-1s also have shorter cilia compared to HUVECs (3.0 ± 1.0 μm versus 5.1 ± 2.4 μm, at confluence, p = 0.003).

Conclusions: We demonstrate that FBS plays a role in determining the prevalence of cilia in HUVECs. In doing so, we highlight the importance of considering a commonly varied parameter (% FBS), in the experimental design. We recommend that future studies examining large blood vessel EC primary cilia use confluent HUVECs grown in high serum medium, as we found these cells to have a higher cilia incidence than low serum media HUVECs. For studies interested in microvasculature EC primary cilia, we recommend using cobblestone HMEC-1s grown in high serum medium, as these cells have a 19.5 ± 6.2 % cilia incidence.

No MeSH data available.


Use of co-labelling to identify primary cilia HUVEC and HMEC-1 primary cilia identification using both axonemal marker arl13b (a, b) and acetylated -tubulin marker 611b (a′, b′, white arrows). (a″, b″) Co-localisation of both antibodies indicates presence of cilium. a-a′) HUVECs grown in high serum (20 % FBS) confluent. b, b″ HMEC-1s are grown in low serum (2 % FBS) then serum-starved (0 % FBS for 48 h)
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Fig4: Use of co-labelling to identify primary cilia HUVEC and HMEC-1 primary cilia identification using both axonemal marker arl13b (a, b) and acetylated -tubulin marker 611b (a′, b′, white arrows). (a″, b″) Co-localisation of both antibodies indicates presence of cilium. a-a′) HUVECs grown in high serum (20 % FBS) confluent. b, b″ HMEC-1s are grown in low serum (2 % FBS) then serum-starved (0 % FBS for 48 h)

Mentions: Using a combination of arl13b and acetylated -tubulin positive labelling, primary cilia were identified on both HUVECs and HMEC-1s (Fig. 4), with greatest incidence of 19.5 ± 6.2 % (median ± quartile range) in HMEC-1s expanded in high serum conditions followed by culture in high serum media for 5 days.


Culture and detection of primary cilia in endothelial cell models.

Lim YC, McGlashan SR, Cooling MT, Long DS - Cilia (2015)

Use of co-labelling to identify primary cilia HUVEC and HMEC-1 primary cilia identification using both axonemal marker arl13b (a, b) and acetylated -tubulin marker 611b (a′, b′, white arrows). (a″, b″) Co-localisation of both antibodies indicates presence of cilium. a-a′) HUVECs grown in high serum (20 % FBS) confluent. b, b″ HMEC-1s are grown in low serum (2 % FBS) then serum-starved (0 % FBS for 48 h)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4590708&req=5

Fig4: Use of co-labelling to identify primary cilia HUVEC and HMEC-1 primary cilia identification using both axonemal marker arl13b (a, b) and acetylated -tubulin marker 611b (a′, b′, white arrows). (a″, b″) Co-localisation of both antibodies indicates presence of cilium. a-a′) HUVECs grown in high serum (20 % FBS) confluent. b, b″ HMEC-1s are grown in low serum (2 % FBS) then serum-starved (0 % FBS for 48 h)
Mentions: Using a combination of arl13b and acetylated -tubulin positive labelling, primary cilia were identified on both HUVECs and HMEC-1s (Fig. 4), with greatest incidence of 19.5 ± 6.2 % (median ± quartile range) in HMEC-1s expanded in high serum conditions followed by culture in high serum media for 5 days.

Bottom Line: HMEC-1s also have shorter cilia compared to HUVECs (3.0 ± 1.0 μm versus 5.1 ± 2.4 μm, at confluence, p = 0.003).We recommend that future studies examining large blood vessel EC primary cilia use confluent HUVECs grown in high serum medium, as we found these cells to have a higher cilia incidence than low serum media HUVECs.For studies interested in microvasculature EC primary cilia, we recommend using cobblestone HMEC-1s grown in high serum medium, as these cells have a 19.5 ± 6.2 % cilia incidence.

View Article: PubMed Central - PubMed

Affiliation: Auckland Bioengineering Institute, University of Auckland, 70 Symonds Street, Auckland, 1142 New Zealand.

ABSTRACT

Background: The primary cilium is a sensor of blood-induced forces in endothelial cells (ECs). Studies that have examined EC primary cilia have reported a wide range of cilia incidence (percentage of ciliated cells). We hypothesise that this variation is due to the diversity in culture conditions in which the cells are grown. We studied two EC types: human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMEC-1s). Both cell types were grown in media containing foetal bovine serum (FBS) at high (20 % FBS and 10 % FBS for HUVECs and HMEC-1s, respectively) or low (2 % FBS) concentrations. Cells were then either fixed at confluence, serum-starved or grown post-confluence for 5 days in corresponding expansion media (cobblestone treatment). For each culture condition, we quantified cilia incidence and length.

Results: HUVEC ciliogenesis is dependent on serum concentration during the growth phase; low serum (2 % FBS) HUVECs were not ciliated, whereas high serum (20 % FBS) confluent HUVECs have a cilia incidence of 2.1 ± 2.2 % (median ± interquartile range). We report, for the first time, the presence of cilia in the HMEC-1 cell type. HMEC-1s have between 2.2 and 3.5 times greater cilia incidence than HUVECs (p < 0.001). HMEC-1s also have shorter cilia compared to HUVECs (3.0 ± 1.0 μm versus 5.1 ± 2.4 μm, at confluence, p = 0.003).

Conclusions: We demonstrate that FBS plays a role in determining the prevalence of cilia in HUVECs. In doing so, we highlight the importance of considering a commonly varied parameter (% FBS), in the experimental design. We recommend that future studies examining large blood vessel EC primary cilia use confluent HUVECs grown in high serum medium, as we found these cells to have a higher cilia incidence than low serum media HUVECs. For studies interested in microvasculature EC primary cilia, we recommend using cobblestone HMEC-1s grown in high serum medium, as these cells have a 19.5 ± 6.2 % cilia incidence.

No MeSH data available.