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Essay: Sphingolipids

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Summary

In this project is the effect of cholesterol at ceramide converting in sphingomyeline, glucosylceramide, ceramide 1-fosfaat and sphingosine in SK-N-AS and HeLa cells discovered. Those sphingolipids are found in mammalian cell membranes are involved in a variety of different functions like first/second messengers, membrane lipid rafts, and in a lot of different signalling pathways (See figure 2)

Cholesterol is also an important part of the mammalian cell membrane. It is involved in maintenance and stability of the membrane and in synthase of important molecules like vitamin D and steroid hormones. Cholesterol is synthesised by the liver but is also a present in some nutrition. Cholesterol and sphingomyelin have a high affinity for each other, because of van der Waals interactions. Recent studies show the effect of cholesterol at sphingomyelin synthase. SMase activity showed a strongly negative correlation between SMase activity and the Cholesterol/Protein ratio.

Two types of tumour cells will be tested SK-N- AS which are from a patient with neuroblastoma. And HeLa cells which are from a patient with cervical carcinoma. When sphingolipid metabolism is dysregulated, sphingolipid metabolism is associated with the pathogenesis and development of various types of cancers. Sphingosine 1-fosfaat is influencing cell growth in neuroblastoma cells. Sphingomyeline is influencing cell growth in cervical carcinoma cells.

Introduction

Ceramide is converting into other sphingolipids like sphingomyelin, sphingoceramide and sphingosine. It is known that sphingomyelin synthase is affected by cholesterol. In this project the effect of cholesterol at the converting of ceramide in sphingomyelin, sphingoceramide and sphingosine in SKNAS and HELA cells is examined. Cholesterol in the cells will be decreased by exposing cells to methyl-beta-cyclodextrin and will be increased by exposing cells to cholesterol-methyl-beta-cyclodextrin inclusion complexes. Ceramide will be coloured with C6-NBD. With thin-layer chromatography can be analysed in which sphingolipids ceramide is converted. With protein-analyses can be determined the amount of cells each wells so the samples can be compared to each other.

Main question what is the effect of cholesterol at converting ceramide in sphingomyelin, glucosylceramide, ceramide 1-phosphate and sphingosine in SKNAS and HELA cells?

Hypothesis: Sphingomyelin synthase will be influenced by cholesterol. More cholesterol means more sphingomyelin. Cholesterol has no effect at the synthase of other sphingolipids. HeLa cells are more influenced by cholesterol than SK-N-AS cells.

Sphingolipids

The sphingolipids are named by J.L.W.Thudichum in 1884, because of their mysteriousness and because Thudichum was a fan of the sphinx from Greek mythology, he named it after the sphinx.

Sphingolipids are a form of lipids (fat like molecules often but not exclusively seen in cell membranes) which are characterized by their backbone, consisting of eighteen carbon amino alcohol bases. They are synthesized in the Endoplasmic reticulum from non-sphingolipid precursors. This family of lipids play an important role in membrane biology and is involved in various different cell signalling pathways. (Gault CR, 2010)

The three prime lipid classes (glycerolipids, sphingolipids and sterols) in animal cells membranes are widely known. But most don’t realize how many combinations there are in the fundamental structure in each lipid class, which is relevant in the specialization of the lipid. There were scientist who believed sphingolipids mainly existed to keep the cell membrane stable, but some studies have elevated these sphingolipids to play a meaningful part in some biological mechanisms. Furthermore there are 10^9 lipid molecules in a small animal cell. This begs the question what exactly are the functions of sphingolipids?

Pretty much all membrane lipids are amphipathic (which means they are both hydrophilic and hydrophobic) The hydrophilic region is made up of phosphate groups, sugar residues, and/or hydroxyl groups. And the hydrophobic part consist of a long-chain base (sphingoid base) which can have two or even three hydroxyl groups. (Anthony, 2004)

Figure 1: structure of some sphingolipids, in blue only one kind of sphingoid base (sphingosine),in red only one kind of fatty acid (palmitic acid). (Anthony, 2004)

Functions of sphingolipids

Sphingolipids are known to act as both first and second messengers in several signalling pathways. First messengers are extracellular factors like hormones or neurotransmitters that can trigger biological effects in the cell like growth immune responses etc. Second messengers are intracellular signalling molecules released by the cell to trigger biological effects such as proliferation, differentiation etc. (figure 2)

Figure 2: Different participation of sphingolipids in cell biology signalling. (Obeid, 2008)

They also play an important role in the membrane lipid rafts. These are plasma membranes containing combinations of glycosphingolipids and protein receptors organized in glycolipoprotein microdomains. Which function as centres for protein sorting and signal transduction. In these rafts sphingomyelin levels are increased by about half in comparison to plasma membranes. (Pike, 2009)

Metabolic pathway of sphingolipids

In the sphingolipid metabolic pathway ceramide is at the centre of it all being synthesized from the molecules palmitate and serine (de novo which means newly made from simple molecules), which reduces to form 3-keto-dihydrosphingosine, which reduces to dihydrosphingosine, dihydrosphingosine is acetylated trough dihydro-ceramide synthase (CerS), and becomes dihydroceramide, followed by desaturase into finally ceramide.

It can also form by sphingomyelinase of dihydrosphingomyelin to dihydroceramide followed by desaturase to ceramide, and by sphingomyelinase of sphingomyelin to ceramide. This last one can go both ways ceramide can become sphingomyelin through sphingomyelin synthase (SMS).

It can form through glucosylceramidase (GCase) from glucosylceramide, and again both ways through glucosylceramide synthase (GCS).

Ceramide can form from ceramide 1-phospate with phosphatase and back again through a specific ceramide kinase (CK).
And lastly it can form from sphingosine 1-phosphate through sphingosine-1-phosphate phosphatase (SPPase) it becomes sphingosine which in turn becomes ceramide with ceramide synthase (CerS)

Figure 3: Sphingolipids metabolism with their related enzymes. (Obeid, 2008)

Ceramide

Ceramide is one of the plainest sphingolipid as seen in figure 1 it is only composed of a sphingosine and a fatty acid. It has been suggested that the fatty acid can largely decide the function and pathway of this type of sphingolipid. Which fatty acid is N-acylated to the long chain-base is being decided through various genes so they are responsible for several distinct ceramide groups. The distribution of these genes to various tissues suggests some tissues need precise behaviour from these ceramide. The main pattern of these ceramide may be portrayed, but some factors are still unclear of these sphingolipids, like how the specific enzymes are regulated and how many separate enzymes can influence the same pathway.

Ceramide is synthesized in the endoplasmic reticulum, it uses the cells own components like vesicular transport but also different transport methods to move from the endoplasmic reticulum to the golgi apparatus. It uses these methods because it can’t move through the cytosol by itself since it has hydrophobic properties. (Anthony, 2004)

How can ceramide synthesis be qualified?

Ceramide synthesis can be qualified by using NBD C6-Ceramide (6-((N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)amino)hexanoyl)Sphingosine) which will emit a green fluorescence. Its absorption maxima is 466nm and its emitting maxima is 536nm. There are several experiments to qualify ceramide synthesis like:

  • TLC, (thin layer chromatography)
  • HPLC, (High-performance liquid chromatography)
  • Mass spectrometry, (Cremesti, 2000)

Glucosylceramide

Glucosylceramide is a more complex sphingolipid, it has a carbohydrate head group which is attached to the 1-hydroxy group of ceramide. The most simple glucosylceramide are galactosylceramide and glucosylceramide. At which galactosylceramide has a glucose group, and glucosylceramide a galactose group. From these glucosylceramide more complex sphingolipids can be synthesized through adding additional glycose sub-units. (Obeid, 2008)

Ceramide 1-fosfaat

Ceramide-1-phosphate is formed from ceramide by a specific ceramide kinase (CK) as stated in ‘What is the metabolic pathway of sphingolipids?’. Thus far the single enzyme known to produce Ceramide-1-phosphate in mammalian cells is CK.
This ceramide is involved in the mitogenesis (induction of mitosis) of certain cells, as well as having antiapoptic effects. Ceramide-1-phosphate is involved in inflammatory reactions also. The effects of Ceramide-1-phosphate are mostly active in intracellular compartments. (G”mez-Mundoz, 2010)

Sphingomyelin

Sphingomyelin is named by the myelin sheets around nerve cells. Sphingomyelin is mostly found in myelin sheets, but is also found in other cell membranes. The only phospholipid which is also a sphingolipid is sphingomyelin. One of the important nerve cell membrane components is sphingomyelin. Sphingomyelin synthase(SMS) catalyses the transfer of phosphoryl choline from phosphatidylcholine to a ceramide. SMS1 and SMS2 are the human SMS genes.

The SMS1 gene is find in the trans-Golgi apparatus is encoded. the SMS 2 gene is associated with the plasma membrane.

Figure 4. synthase and degradation of sphingomyelin by the enzymes sphingomyelin synthase(SMS) and sphingomyelinase (ASMase/SMase)

Sphingomyelinase causes the release of ceramide and phosphocholine. Sphingomyelin is converted to ceramide because of this reaction. Sphingomyelinase is also called ASMase or aSMase because sphingomyelinase functions at acidic pH.
The synthase of sphingomyelinase is seen in figure 4. (King(pHd), 2016)

Sphingomyelin has a few different functions. Myelin sheets around nerve cells has a lot of sphingomyelins. This suggests their function as insulator of nerve fibers. (Voet, Voet, & Pratt, 2008) Sphingomyelin is also found in the plasma membranes of other cells. Sphingomyelin is in the plasma membrane also important for entering iron into cells. Sphingomyelin has also a function in the activity of some membrane-bound proteins including a certain receptors and ion channels. The most important sphingolipid in the nucleus is sphingomyelin because it is involved in chromatin dynamics. (Christie, 2014)

Sphingosine

The initiation of sphingosine synthesis takes places via condensation of serine and palmitoyl-CoA. This reaction is catalysed by the enzyme serine palmitodyltransferase(SPT). Hereby is 3-ketosphinganine(3-ketodihydrosphingosine) formed. SPT contains two main subunits. SPTLC1 and SPTLC2. The isoform of SPTLC2L isoform is also called SPTLC3. SPTLC1 is found in active SPT enzymes. Some tissues contains SPTLC2 subunits, and other contains SPTLC3 subunits. LC means Long-Chain subunit. 3-ketosphinganine is converted into sphinganine (dihydrosphingosine) by 3-ketosphinganine reductase. Sphinganine is converted into dihydroceramide by the enzyme ceramide synthase. Dihydroceramide is converted into ceramide by the enzyme dihydroceramide.
Through the action of ceramide synthases and ceramidases, sphingosine has a function as substrate for ceramide synthesis, and ceramide has a function as substrate for sphingosine synthesis. (King(pHd), 2016)
The synthase of sphingosine is shown in figure 5

Figure 5. the synthase of sphingosine. The initiation of sphingosinesynthese starts with condensation of serine and palmitoyl-CoA. The synthase continuous by the help of 3-ketosphinganine reductase, ceramide synthase, dihydroceramide desaturase and ceramidase. (King(pHd), 2016)

Sphingosine is converted into sphingosine 1-phosphaat via sphingosine kinase. Sphingosine 1-phosphaat can be converted in sphingosine via different phosphatases.

Sphingosine 1-phosphate (S1P) is released to the extracellular space. After that, it binds to specific receptors on the plasma membrane of target cells. S1P has important roles in differentiation, migration and cell proliferation. (Tsuyoshi Nishia, 2013) Because of its role in cell differentiation, S1P also has a role in cell differentiation in cancer cells.

SK-N-AS and HeLa cells

In this experiment two types tumour cells are tested. SK-N-AS cells are originally from a female patient with neuroblastoma from the metastasis of the bone marrow. (Aldrich, 2016) Neuroblastoma is a tumour cancer which is mostly located in the nerve tissue of the adrenal gland but is also found in nerve tissues in other body parts. Approximately 15 percent of all childhood cancer deaths is caused by neuroblastoma. (Mehrdad Rahmaniyan, 2012)

When sphingolipid metabolism is dysregulated, sphingolipid metabolism is associated with the pathogenesis and development of various types of cancers.

In neuroblastoma cells and tissues is sphingosine kinase 2 is highly expressed. Spingosine-1-phosphaat(SIP) is the product of sphingosine kinase 2. Vascular endothelial growth factor (VEGF) expression is induced by spingosine-1-phosphaat. VEGF is a factor who is regulating the process of angiogenesis. Angiogenesis is an essential factor for metastasis and tumour growth. VEGF expression is induced via HIF-1-” independent pathway. Spingosine-1-phosphate receptor 2(S1P2) correlates with VEGF mRNA expression. This suggest that the VEGF/S1P/S1P2 pathway may promote neuroblastoma growth. (Mehrdad Rahmaniyan, 2012)
HeLa cells are originally from a 31 year old female with cervical carcinoma. (Aldrich s. , 2016) Sphingomyelin synthase 1 and sphingomyelin synthase 2 are co-expressed and function as golgi-and plasma membrane-associated SM synthase in hum cervia carcinoma cells. RNA interference-mediated decrease of sphingomyelin synthase-1 and sphingomyelin synthase-2 caused sphingomyelin production, accumulation of ceramide and a block in cell growth. (Fikadu Geta Tafesse, 2007)

Cholesterol

All human cells have plasma membranes. The fundamental structure is the phospholipid bilayer. Exist the phospholipid bilayer, the plasma membrane consists lipids and proteins. Another essential compound of mammalian plasma membranes is cholesterol.
Cholesterol is responsible for maintenance of cell structure and maintaining the stability and the stiffness of the plasma membrane. 30% of the cell membranes exits of cholesterol. (Groningen Biomolecular Sciences and Biotechnology Institute en het Zernike Institute for Advanced Materials van de Rijksuniversiteit Groningen, 2014)

Cholesterol is also a precursor of compounds as vitamin D, steroid hormones and bile acid Cholesterol has four rings in its structure as seen in figure 6.

Cholesterol is synthesized in the liver by mammalians self, but is also a part of some food. The cholesterol in the diet, is mostly find in eggs, meat, cheese and other dairy products. Normally 30-60% of the cholesterol in the western diet(approximately 500 mg) is absorbed by the gut. Cholesterol is a lipid molecule so it is poorly soluble in water. Lipoproteins transport cholesterol between organs and tissues. The sterol ring of cholesterol cannot be metabolized by humans, it is excreted as bile acids or as free cholesterol. Approximately 50% of the cholesterol eliminated from the body through faeces each day is excreted as bile acids and the remainder as the product of bacterial reduction of free cholesterol in the gut. The two sources of cholesterol reach cells in different ways. The intracellular free cholesterol can be newly synthesized within the cell but can also be derived from lipoproteins. the exogenous cholesterol can only be derived from lipoproteins.

Lipoproteins

Cells are taken up the exogenous(dietary) cholesterol in lipoproteins. Cholesterol is an element of lipoproteins. Lipoproteins transport fats in the body and consist of cholesterol, triglycerides, proteins and phospholipids. There are five different types of lipoproteins: Very Low Density Lipoproteins (VLDL), Low Density Lipoproteins (LDL), Intermediate Density Lipoproteins (IDL), High Density Lipoproteins (HDL) and chylomicrons. (Dominiczak, 2009)

The types of lipoproteins vary in density, apolipoprotein composition and in the amounts of phospholipids, cholesteryl esters, triglycerides, proteins and free cholesterol. The differences are seen in table 1.
chylomicron VLDL IDL LDL HDL

Density (g/ml) <0.95 0.950’1.006 1.006’1.019 1.019’1.063 1.063’1.210
Components (% dry weight)
protein 2 7 15 20 40’55
triglycerides 83 50 31 10 8
free cholesterol 2 7 7 8 4
cholesteryl esters 3 12 23 42 12’20
phospholipids 7 20 22 22 22
Apoprotein composition A-I, A-II,
B-48, C-I,
C-II, C-III B-100, C-I,
C-II, C-III,
E B-100, C-I,
C-II, C-III,
E B-100 A-I, A-II,
C-I, C-II,
C-III, D, E
Table 1. (Christopher K Mathews, 2000)

Chylomicrons

Chylomicrons consists approximately of proteins(1-2%), cholesterol(1-3%) phospholipids(6-12%) and triglycerides(85-92%). (Hussain, 2000) Their function is transport of fats absorbed by the intestinal epithelial cells by the lymphatic system and the blood to the rest of the body. Dietary fats are digested and have a role in forming the chylomicrons’ triglycerides, free cholesterol and cholesteryl esters. (Thompson, 2015)

Very low density lipoproteins(VLDL) and intermediate density lipoproteins(IDL)

The synthase of VLDL’s is placed in the liver. VLDL transports triacylglycerol to the tissue cells. Triacylglycerol’s are synthesized in the liver. VLDL is hydrolase by lipoprotein lipase on the same way as chylomicrons. VLDL is now called IDL. IDL will be hydrolyzed and transformed into LDL or will be taken up by the liver.

Low density lipoproteins(LDL)

Apolipoprotein B100 is the only apolipoprotein of LDL and binds lipoprotein particles to LDL-specific receptors. VLDL and IDL have also apolipoprotein B100 but they also contain other apolipoproteins. cholesterol in LDL is converted to steroid hormones or is used as structural component of cell membranes.

High density lipoproteins(HDL)

The excess of cholesterol in cells is removed and is returned to the liver. HDL has an important role in this process. The cholesterol which has entered the liver is metabolized to salts and bile acids and eliminated by the intestine. HDL and LDL contain together the balance of cholesterol in the body. (Thompson, 2015)

Cholesterol is converted to cholesteryl esters by HDL. This converting takes place by the enzyme LCAT which is activated by apoA-1 in HDL. Cholesteryl esters are transferred to VLDL and LDL by the apo-D protein in HDL. Apo-CII and apo-E proteins are transferred to chylomicrons and other LDL’s. Afterwards apo-E is recognised by the liver, so the remnants of lipoproteins and cholesterol can be converted to bile acids and will be excreted into the duodenum. (Dominiczak, 2009) (Mathews, 2000) (Zamora, 2016)

Decreasing of cholesterol levels

LDL enters the vascular wall very easy. The immune system takes up the lipoproteins, which get overloaded with lipids. The lipoproteins change into foam cells. When foam cells die, they release the accumulated lipids. Those lipids form pools within the vascular wall. As a result plaques arise in the vascular walls. This is called arteriosclerosis. (Dominiczak, 2009) Arteriosclerosis can lead to brain and hart infarcts and transient ischemic attacks(TIA). (slagaderverkalking, 2016)

People with too much cholesterol in their blood, can use medication to decrease the amounts of cholesterol. There are different types of medication to decrease cholesterol in the blood. Statins are medication to decrease cholesterol synthesis by the liver. Less LDL in the blood, decrease the risk of arteriosclerosis. In the experiment the effect of cholesterol at the ceramide converting in SK-N-AS and HeLa will be tested. Statins are not an effective way to decrease cholesterol in SKNAS and HeLa cells, because statins only affect the cholesterol synthase in the liver. To decrease cholesterol in SK-N-AS and HeLA cells , cells are exposed to methyl-beta-cyclodextrin. To increase cellular cholesterol SK-N-AS and HeLa cells, cells are exposed to cholesterol-methyl-beta-cyclodextrin inclusion complexes.

The effect of cholesterol at sphingomyelin synthase

Because of van der Waals interactions, Sphingomyelin and cholesterol a high affinity for each other. They are mostly located together in ‘rafts’ of sub-domains of membranes. There is evidence that sphingomyelin controls the distribution of cholesterol in cells. (Christie, 2014) In a study supplied by M.N. Nikolova-Karakashian, H. Petkova, K.S. Koumanov from the Bulgarian academy of sciences in Sophia, the link between cholesterol and sphingomyeline synthase in rat liver plasma membranes is discovered. It is proved that the SMase activity showed a strongly negative correlation between SMase activity and the Cholesterol/Protein ratio. The enzymes PC:Cer-Pch and PE: Cer-Pet transferase are sphingomyelin synthesising enzymes. Those enzymes were stimulated by increasing cholesterol in feeding. Those results support the link between cholesterol and sphingomyelin synthase. (M.N. Nikolova-Karakashian, 1992)

Conclusion

Sphingolipids are a part of the cell membrane. They have among others a role in cell differentiation and cell growth. Ceramide can be converted into other sphingolipids like sphingosine, sphingomyelin, glucosylceramide and ceramide 1-fosfate. Sphingosine 1-fosfaat affects cell growth in neuroblastoma cells(SK-N-AS). Sphingomyelin affects cell growth in cervical carcinoma cells(HeLa).
Cholesterol and sphingomyelin have a high affinity for each other, because of van der Waals interactions. Recent studies show the effect of cholesterol at sphingomyelin synthase. SMase activity showed a strongly negative correlation between SMase activity and the Cholesterol/Protein ratio. In the practical part of this research, the effect of cholesterol at the ceramide converting in other sphingolipids is discovered.

Discussion

Recent studies showed the effect of cholesterol at sphingomyelin synthase. The cells used in this study are liver cells of a rat. It is not completely sure if the effect of cholesterol at sphingomyelin synthase is the same in SK-N-AS and HeLa cells. If the practical part of this project shows the same results as this study, we still don’t know if the effect works at the same way, or that just the end results are the same

References

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Appendix 1. ‘ Plan of action

The Main question is: What is the effect of cholesterol at converting ceramide in sphingomyelin, glucosylceramide, ceramide-1 phosphate and sphingosine in SKNAS and HELA cells?

The cell lines SKNAS and HELA will be used. which are both genetically unmodified.

Several to be determined concentrations of cholesterol will be tested on these cells, after which ceramide, sphingomyelin, glucosylceramide, ceramide-1 phosphate and sphingosine in these cells will be qualified with TLC (thin layer chromatography), and fluorescence microscopy. Protein determination will also be used to determine whether the amount of cells per well will be accurate.

The cells will be depleted and saturated using Methyl-B-Cyclodextrin wihout cholesterol and with cholesterol respectively.

The sphingolipids: ceramide, sphingomyelin, glucosylceramide, ceramide-1 phosphate and sphingosine will be made fluorescence by NBD C6-Ceramide (6-((N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)amino)hexanoyl)Sphingosine).

Needed protocols:

1-PROTOCOL – CELL CULTURE

Goal: -Starting the experiment with the right kind/amount of cells and wells with the right medium.
Principle: Cells need nutrition and favourable conditions to grow.

2-PROTOCOL – EXPOSURE OF CULTURED CELLS TO C6-NBD-CERAMIDE

Goal: -By adding c6NBD-ceramide the lipids will be fluorescent so they can be qualified.
Principle:c6NBD-ceramide will be green fluorescent after being excited by 466 nm.

3-PROTOCOL ‘ Experimental,

‘The rate of sphingomyelin synthesis de novo is influenced by the level of cholesterol in cultured human skin ‘broblasts’ (adding cholesterol)

Goal: -Adding and removing cholesterol from the SKNAS and HeLa cell lines.
Principle: Methyl-B-cyclodextrin has a central cavity in which cholesterol can be incapsulated.

4-PROTOCOL – HARVESTING OF CULTURED CELLS

Goal: -Seeing the cells through an fluorescence microscope ‘ Getting the cells out their wells and into Eppendorf vials.
Principle:c6NBD-ceramide will be green fluorescent after being excited by 466 nm.

5-PROTOCOL – TOTAL PROTEIN QUANTIFICATION

Goal: -Determine how much proteins are in each well, so qualification can be accurately adjusted to the amount of cells.
Principle: -Each cell has a certain amount of proteins so if one well has 20% less proteins than the other wells it most likely had 20% less cells as well.

6-PROTOCOL – DISRUPTION AND HOMOGENIZATION OF CELLS

Goal:-Getting the lipids in the cell membranes loose.
Principle:-Sonication uses sound energy to agitate particles in a solution.

7-PROTOCOL – TWO-PHASE LIPID EXTRACTION

Goal: -Extract the lipids from the homogenized solution
Principle:- Dichloromethane is widely used as an organic solvent, after centrifugation it will go to the bottom with all lipids.

8-PROTOCOL – LIPID SEPARATION BY THIN LAYER CHROMATOGRAPHY (TLC)

Goal: Separating the different sphingolipids.
Principle: The separate lipids each have different solubility in the solvent and different attraction to the stationary phase, so they will travel at different speeds and therefor separate.

9-PROTOCOL – PLATE IMAGING AND SPOT QUANTIFICATION

Goal:-Viewing the lipid bands on the TLC plate.
Principle:c6NBD-ceramide will be green fluorescent after being excited by 466 nm.

10-PROTOCOL – SILICA EXTRACTION OF NBD-LABELED LIPIDS

Goal:-Getting a more accurate reading on the amount of NBD-labelled lipids.
Principle:-The lipids are separated on the TLC plate so if you scrape it off and measure it using a plate reader it will give the amount of fluorescence and so information on the individual amount of NBD-labelled lipids.

Design of experiment:

Day 1.1: Washing cells with Methyl-B-Cyclodextrin (Depleting cells of cholesterol)
Day 1.2: Adding NBD C6-Ceramide (Colouring sphingolipids)
Day 1.3: Adding cholesterol using Methyl-B-Cyclodextrin+Cholesterol (saturating cells with cholesterol)
Day 2?.1: Harvesting cultured cells , fluorescence microscopy
Day 2?.2: Disruption and homogenization of cells
Day 2?.3: Two-phase lipid extraction
Day 2?.4: Total protein quantification
Day 3?.1: Lipid separation by Thin layer chromatography
Day 3?.2: Plate imaging of TLC plate
Day 3?.3: Extraction of NBD-labeled lipids from the TLC plate ‘ fluorescence measurement

Tabel 1: Cholesterol concentrations in SKNAS and HeLa cell lines (duplo)
Cholesterol (ug) untreated Only washed x x x x
1SKNAS(ml) 2 2 2 2 2 2
2SKNAS(ml) 2 2 2 2 2 2
3HeLa(ml) 2 2 2 2 2 2
4HeLa(ml) 2 2 2 2 2 2

Requirements materials and equipment per protocol:

1)

  • tissue culture cabinet
  • phosphate buffered saline (PBS)
  • trypsin-EDTA solution
  • DMEM (5% FCS), including penicillin/streptomycin
  • sterile blue-capped 15 ml tubes
  • centrifuge
  • p1000 pipette
  • sterile 1000 ”l pipette tips
  • sterile 25 cm2 cell culture flasks
  • incubator
  • phase contrast microscope

2)

  • Cell culture facilities (flow cabinet, incubator, sterile pipettes etc.)
  • Phosphate buffered saline (PBS)
  • DMEM (5% FCS) (including penicillin/streptomycin)
  • 1 mM stock solution of C6-NBD-ceramide in ethanol Preparations by students
  • Cultured cells in 25 cm2 flasks at 40 to 70% confluence. (HeLa,SKNAS)

3)

  • Methyl-B-cyclodextrin (10 mg)
  • Cholesterol (25 mg)
  • 50 ml tube (6)
  • Medium DMEM+p/s+MEM-NEAA+fbs 10% (60 ml)

4)

  • phosphate-buffered saline (PBS)
  • icebox with ice
  • cell scraper
  • 15 ml tubes
  • 2 ml vials
  • Eppendorf centrifuge with cooling unit

5)

  • bicinchoninic acid solution (BCA)
  • 4% (w/v) copper (II) sulphate solution
  • bovine serum albumin (BSA) solution (1 mg/ml)
  • BioRad model 680 spectrophotometric plate reader

6)

  • sonifier (Hielscher UP100H)
  • Dounce
  • Ice box with ice
  • Pre-cooled phosphate-buffered saline (PBS)

7)

  • p1000 pipette
  • vortex
  • Eppendorf centrifuge
  • sample concentrator
  • nitrogen (10 liter bottle), including an adjustable pressure reducer
  • waste tank for organic solvents
  • dichloromethane (DCM)
  • methanol (MeOH)
  • DCM/MeOH (1:2 by vol.)
  • Millipore water (MQ)

8)

  • TLC tank (including lid)
  • filter paper
  • glass cylinder with measuring units
  • dichloromethane (CH2Cl2)
  • methanol (CH3OH)
  • 25% ammonia (NH4OH)
  • glass TLC plates (20 x 10 cm kieselgel 60) – Merck # 1.95626.0001
  • soft carbon pencil (4B)
  • vortex
  • p20 and p200 pipettes
  • extra fine p20 tips
  • hair dryer
  • UV tray
  • iodine grains
  • liquid waste tank for halogen-containing organic solvents.
  • Solvent mix: dichloromethane (CH2Cl2) / methanol (CH3OH) / 25% ammonia (NH4OH) (60:25:1, by volume). Preparations (by students)

9)

  • BioRad ChemiDoc XRS+ Molecular Imager station

10)

  • Soft carbon pencil (4B)
  • 1.5 ml Eppendorf vials
  • Vortex
  • Aluminum foil
  • Curved surgical blades + holders
  • Black flat-bottom microtiter plates
  • 70% ethanol

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