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Chylomicrons produced by Caco-2 cells contained ApoB-48 with diameter of 80-200 nm.

Nauli AM, Sun Y, Whittimore JD, Atyia S, Krishnaswamy G, Nauli SM - Physiol Rep (2014)

Bottom Line: We then tested the effects of cell differentiation, oleic acid, mono-olein, egg lecithin, incubation time, and collagen matrix on CM secretion.We found that cell differentiation, oleic acid, and lecithin were critical for CM secretion.In conclusion, when fully differentiated Caco-2 were challenged with oleic acid, lecithin, and sodium taurocholate, 21% of their total number of lipoproteins were CMs with the diameter of 80-200 nm.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, California Northstate University, Elk Grove, California.

No MeSH data available.


Related in: MedlinePlus

Fat Red 7B staining of the lipoprotein layers separated by sequential NaCl density gradient ultracentrifugation. (A) Thirteen‐day postconfluent cells were incubated for 4 h with 2 mmol/L OA, 1.36 mmol/L lecithin, and 1 mmol/L NaTC in growth media. The secreted lipoproteins were then collected for 2 h in colorless growth media and were stained with Fat Red 7B. The density of the prestained media was adjusted to 1.2 g/mL with NaCl and was carefully overlaid with 500 μL of water. After a 30‐min spin at 10,000 rpm, the top ~500 μL CM layer became heavily stained and the middle layer was lightly stained. The top 500‐μL CM layer was immediately replaced with water and subjected to a 24‐h spin at 65,000 rpm. Notice that after the second spin, the top ~500 μL VLDL layer and the bottom layer were heavily stained and the middle layer became clear. (B) Similar staining and spinning were performed, except with media without lipoproteins. The top low‐density layer was not stained at the end of each spin.
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fig04: Fat Red 7B staining of the lipoprotein layers separated by sequential NaCl density gradient ultracentrifugation. (A) Thirteen‐day postconfluent cells were incubated for 4 h with 2 mmol/L OA, 1.36 mmol/L lecithin, and 1 mmol/L NaTC in growth media. The secreted lipoproteins were then collected for 2 h in colorless growth media and were stained with Fat Red 7B. The density of the prestained media was adjusted to 1.2 g/mL with NaCl and was carefully overlaid with 500 μL of water. After a 30‐min spin at 10,000 rpm, the top ~500 μL CM layer became heavily stained and the middle layer was lightly stained. The top 500‐μL CM layer was immediately replaced with water and subjected to a 24‐h spin at 65,000 rpm. Notice that after the second spin, the top ~500 μL VLDL layer and the bottom layer were heavily stained and the middle layer became clear. (B) Similar staining and spinning were performed, except with media without lipoproteins. The top low‐density layer was not stained at the end of each spin.

Mentions: Caco‐2, human epithelial colorectal adenocarcinoma, cells (passage 17) were obtained from American Type Culture Collection (Manassas, VA) and were grown at 37°C with 5% CO2 in growth media (DMEM with 15% FBS). For propagation purposes, cells were split (1:6) when they have reached 50–70% confluence. Media was changed every other day. For the initial experiments, cells (passage 40–60) were grown in 10‐cm tissue culture dishes. For the later experiments, cells were grown on Tranwells (Cat. # 3420, 100‐mm dish, 3‐μm pore size, polycarbonate membrane; Corning, Inc., Tewksbury, MA). The prefiltered lipid mixture (unless specified, OA:lecithin:sodium taurocholate (NaTC) = 2:1.36:1.0 mmol/L) in 10‐mL growth media was added to the cells to induce the secretion of CMs. Cells that were grown in the tissue culture dishes were incubated with lipid mixture for 4 h, washed twice with PBS, and incubated with fresh growth media for 2 h to collect for the secreted lipoproteins. Cells grown on Transwells (Corning, Inc.) were incubated for 4 h with lipid mixture in the apical chamber and growth media without lipid mixture in the basolateral chamber. To isolate the CM and VLDL layers, the lipoprotein‐containing media were subjected to sequential NaCl density gradient ultracentrifugation (see Appendix). The successful isolation of these lipoproteins was confirmed by Fat Red 7B staining (Fig. A1), ApoB, TG, and TEM analysis (Fig. A2).


Chylomicrons produced by Caco-2 cells contained ApoB-48 with diameter of 80-200 nm.

Nauli AM, Sun Y, Whittimore JD, Atyia S, Krishnaswamy G, Nauli SM - Physiol Rep (2014)

Fat Red 7B staining of the lipoprotein layers separated by sequential NaCl density gradient ultracentrifugation. (A) Thirteen‐day postconfluent cells were incubated for 4 h with 2 mmol/L OA, 1.36 mmol/L lecithin, and 1 mmol/L NaTC in growth media. The secreted lipoproteins were then collected for 2 h in colorless growth media and were stained with Fat Red 7B. The density of the prestained media was adjusted to 1.2 g/mL with NaCl and was carefully overlaid with 500 μL of water. After a 30‐min spin at 10,000 rpm, the top ~500 μL CM layer became heavily stained and the middle layer was lightly stained. The top 500‐μL CM layer was immediately replaced with water and subjected to a 24‐h spin at 65,000 rpm. Notice that after the second spin, the top ~500 μL VLDL layer and the bottom layer were heavily stained and the middle layer became clear. (B) Similar staining and spinning were performed, except with media without lipoproteins. The top low‐density layer was not stained at the end of each spin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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fig04: Fat Red 7B staining of the lipoprotein layers separated by sequential NaCl density gradient ultracentrifugation. (A) Thirteen‐day postconfluent cells were incubated for 4 h with 2 mmol/L OA, 1.36 mmol/L lecithin, and 1 mmol/L NaTC in growth media. The secreted lipoproteins were then collected for 2 h in colorless growth media and were stained with Fat Red 7B. The density of the prestained media was adjusted to 1.2 g/mL with NaCl and was carefully overlaid with 500 μL of water. After a 30‐min spin at 10,000 rpm, the top ~500 μL CM layer became heavily stained and the middle layer was lightly stained. The top 500‐μL CM layer was immediately replaced with water and subjected to a 24‐h spin at 65,000 rpm. Notice that after the second spin, the top ~500 μL VLDL layer and the bottom layer were heavily stained and the middle layer became clear. (B) Similar staining and spinning were performed, except with media without lipoproteins. The top low‐density layer was not stained at the end of each spin.
Mentions: Caco‐2, human epithelial colorectal adenocarcinoma, cells (passage 17) were obtained from American Type Culture Collection (Manassas, VA) and were grown at 37°C with 5% CO2 in growth media (DMEM with 15% FBS). For propagation purposes, cells were split (1:6) when they have reached 50–70% confluence. Media was changed every other day. For the initial experiments, cells (passage 40–60) were grown in 10‐cm tissue culture dishes. For the later experiments, cells were grown on Tranwells (Cat. # 3420, 100‐mm dish, 3‐μm pore size, polycarbonate membrane; Corning, Inc., Tewksbury, MA). The prefiltered lipid mixture (unless specified, OA:lecithin:sodium taurocholate (NaTC) = 2:1.36:1.0 mmol/L) in 10‐mL growth media was added to the cells to induce the secretion of CMs. Cells that were grown in the tissue culture dishes were incubated with lipid mixture for 4 h, washed twice with PBS, and incubated with fresh growth media for 2 h to collect for the secreted lipoproteins. Cells grown on Transwells (Corning, Inc.) were incubated for 4 h with lipid mixture in the apical chamber and growth media without lipid mixture in the basolateral chamber. To isolate the CM and VLDL layers, the lipoprotein‐containing media were subjected to sequential NaCl density gradient ultracentrifugation (see Appendix). The successful isolation of these lipoproteins was confirmed by Fat Red 7B staining (Fig. A1), ApoB, TG, and TEM analysis (Fig. A2).

Bottom Line: We then tested the effects of cell differentiation, oleic acid, mono-olein, egg lecithin, incubation time, and collagen matrix on CM secretion.We found that cell differentiation, oleic acid, and lecithin were critical for CM secretion.In conclusion, when fully differentiated Caco-2 were challenged with oleic acid, lecithin, and sodium taurocholate, 21% of their total number of lipoproteins were CMs with the diameter of 80-200 nm.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, California Northstate University, Elk Grove, California.

No MeSH data available.


Related in: MedlinePlus