ab118183 Fatty Acid Oxidation Human Flow Cytometry Kit Instructions for Use For measuring in very long chain specific acyl-coa dehydrogenase, medium-chain specific acyl-coa dehydrogenase and long-chain 3-hydroxyl-CoA dehydrogenase This product is for research use only and is not intended for diagnostic use.
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Table of Contents 1. Introduction 3 2. Assay Summary 5 3. Kit Contents 6 4. Storage and Handling 6 5. Additional Materials Required 7 6. Preparation of Reagents 7 7. Sample Preparation 8 8. Assay Procedure 9 9. Data Analysis 11 10. Assay Performance and Specificity 12 11. Troubleshooting 16 2
1. Introduction Principle: ab118183 is a panel of antibodies against key enzymes of the mitochondrial fatty acid oxidation pathway for flow cytometry. The assay combines the power of single cell analysis obtained from flow cytometry with the specificity of antibody-based immunostaining to quantify protein levels in cultured cells. Cells are harvested and fixed/permeabilized in suspension, targets of interest are detected indirectly with highly specific well-characterized monoclonal antibodies that are then labeled with fluorescent antibodies Background: The Fatty acid B-oxidation (FAO) pathway is a key metabolic pathway that plays an important role in energy homeostasis particularly in organs such as the liver, heart and skeletal muscle. Oxidation of fatty acids occurs inside the mitochondria where acyl-coa esters (activated fatty acids) of various lengths are shortened into units of acetyl-coa each time a cycle is fully completed. Each unit of acetyl-coa is then oxidized by the mitochondria into CO 2 and H 2 O via the citric acid cycle and the mitochondria respiratory chain. The FAO pathway consists of a four step process which involves: (1) dehydrogenation, (2) hydration, (3) second dehydrogenation and (4) thiolytic cleavage. This kit measures the levels of three of the most studied enzymes in this pathway: ACADVL, ACADM, HADHA. These enzymes are not only important in the field of inborn errors of metabolism, but also in metabolic syndrome, type 2-diabetes and compound toxicity. 3
ACADVL is a homodimer that carries out the first step toward esters of long-chain and very long chain fatty acids such as palmitoyl-coa, mysritoyl-coa and stearoyl-coa. It can accommodate substrate acyl chain lengths as long as 24 carbons, but shows little activity for substrates of less than 12 carbons. Defects in ACADVL are the cause of acyl-coa dehydrogenase very long chain deficiency (ACADVLD) [MIM:201475]. It is clinically heterogeneous, with three major phenotypes: (1)cardiomyopathy (2)a hypoketotic hypoglycemia and (3) rhabdomyolysis triggered by exercise or fasting. ACADM is a homotetramer and it also carries out the first step in the pathway but, as opposed to ACADVL, it is specific for acyl chain lengths of 4 to 16 carbons. Defects in ACADM are the cause of acyl- CoA dehydrogenase medium-chain deficiency (ACADMD) [MIM:201450], the most common fatty acid oxidation disorder in humans. This is an autosomal recessive disease which causes fasting hypoglycemia, hepatic dysfunction, and encephalopathy, often resulting in death in infancy. The trifunctional enzyme (TP) is an octomer (4 alpha and 4 beta subunits) that catalyzes three out of the four steps in beta oxidation pathway with a specific affinity for long chain substrates. This kit measures the alpha subunit of the trifunctional protein which catalyzes the hydration of enoyl-coa and the dehydrogenation of 3- hydroxyacyl CoA compounds (steps 2 and 3). Defects in HADHA 4
are a cause of TP deficiency [MIM:609015], long-chain 3-hydroxyl- CoA dehydrogenase deficiency (LCHAD deficiency) [MIM:609016] and maternal acute fatty liver of pregnancy (AFLP) [MIM:609016]. 2. Assay Summary Harvest cells as a single cell suspension. Fix cells with 4% paraformaldehyde for 15 minutes and pellet Permeabilize cells with 1X permeabilization buffer for 15 minutes and pellet Block cells with Blocking/Incubation buffer for 15 minutes and pellet Overlay 2X primary antibodies diluted in Blocking/Incubation buffer; incubate for 1 hour and wash. Add 1X secondary antibodies diluted in Blocking/Incubation buffer, incubate for 1 hour, wash and read with flow cytometer 5
3. Kit Contents Item 10X Phosphate Buffered Saline (PBS) 100X Triton X-100 (10% solution) 400X Tween 20 (20% solution) Blocking Buffer 100X ACADVL Primary antibody 100X ACADM Primary Antibody 100X HADHA Primary Antibody 100X Normal Mouse IgG Quantity 100 ml 1.25 ml 4 ml 25 ml 0.12 ml 0.12 ml 0.12 ml 0.12 ml 4. Storage and Handling Spin down the contents of the antibody vials upon receipt of the kit. Store all components upright at 4 C. This kit is stable for at least 6 months from receipt. 6
5. Additional Materials Required Flow cytometer. Microfuge. 20% paraformaldehyde. Goat anti-mouse detection antibodies labeled with fluorochrome(s) suitable for available flow cytometer (e.g. Abcam, GAM IgG H&L DyLight 488 (ab96871)). 6. Preparation of Reagents 6.1 Prepare 1X PBS by diluting 100 ml of 10X PBS in 900 ml Nanopure water or equivalent. Mix well. Store at room temperature. 6.2 Immediately prior to use prepare sufficient 1X Wash Buffer = 0.05% Tween-20, 1% Blocking buffer in PBS. 6.3 Immediately prior to use prepare enough 1X Permeabilization Buffer = 0.1% Triton X-100 in PBS. 6.4 Immediately prior to use prepare 1X Incubation buffer = 10% blocking solution, 0.1% Triton X-100 in PBS. Any excess should be stored at 4 C for no more than 24 hours. 7
7. Sample Preparation 7.1 Cell culture and treatment conditions are dictated by the experiment at hand. As a general guideline, it is advisable to analyze at least 10,000 events (cells) on the flow cytometer per sample/data point. Therefore at least four times that many cells should be collected per data point to ensure sufficient material at the end of the staining. 7.2 For suspension cells, generate a single cell solution by pipetting up and down. 7.3 For adherent cells, fully dissociate cells (e.g. trypsin) into single cell suspension. Passaging the cell line the day before the experiment onto a fresh cultured plate may help improve single cell dissociation on the day of the experiment. 7.4 Pellet cells at 350 500 x g for 5 minutes (depending on cell size) and aspirate supernatant. Note: It is helpful during the fixation process to leave no more than 100µL of complete media over the cell pellet. 7.5 Resuspend pellet into a suitable volume of PBS to a final concentration of 2 5x10 6 cells per ml. 8
8. Assay Procedure 8.1 Overlay 20% paraformaldehyde such that the final concentration is 4%, gently mix by inverting the tube and incubate at room temperature for 15 minutes. 8.2 Pellet cells at 350-500 x g for 5 minutes (depending on cell size) and decant supernatant. Note: paraformaldehyde is toxic: handle with care and dispose of according to local requirements. 8.3 Dislodge the pellet by gently tapping the bottom of the tube and add 1X Permeabilization buffer to a final concentration of 1 2x10 6 cells per ml. Mix by gently inverting the tube and incubate at room temperature for 15 minutes. 8.4 Pellet cells at 350 500 x g for 5 minutes (depending on cell size) and aspirate supernatant. 8.5 Dislodge the pellet by gently tapping the bottom of the tube and add 50µL of 1X Blocking/Incubation buffer per tube (final concentration is 2x10 4 cells per µl). Mix by gently inverting the tube and incubate at room temperature for 15 minutes. 9
8.6 Prepare 50µL per assay tube of 2X ACADVL Primary Antibody Solution in 1X Blocking/Incubation buffer so that it can be overlayed over the cell suspension for a final 1X antibody solution. 8.7 Repeat previous step for ACADM and HADHA antibodies. Note All antibodies are mouse in origin and should not be mixed. 8.8 Overlay primary antibodies over the cell suspension and mix by gently inverting the tube. Incubate at room temperature for at least 1 hour. 8.9 Pellet cells at 350-500 x g for 5 minutes (depending on cell size) and aspirate supernatant. 8.10 Dislodge the pellet by gently tapping the bottom of the tube and wash twice by centrifugation with 1mL of 1X Wash per assay tube. 8.11 Prepare 100µL of Goat Anti-Mouse detection antibody labeled with a fluorochrome in 1X Blocking/Incubation buffer per assay tube. 8.12 After final wash, dislodge the pellet by gently tapping the bottom of the tube and add 100µL of detector solution per assay tube. Incubate for at least 1 hour at room temperature in the dark. 10
8.13 Pellet cells at 350 500 x g for 5 minutes (depending on cell size) and aspirate supernatant. 8.14 Dislodge the pellet by gently tapping the bottom of the tube and wash twice by centrifugation with 1mL of 1X Wash per assay tube. 8.15 After final wash, dislodge the pellet by gently tapping the bottom of the tube and add 100µL of PBS to each assay tube. 9. Data Analysis Specific methods depend on the available flow cytometer. It is important to appropriately establish forward and side scatter gates to exclude debris and cellular aggregates from analysis. This kit provides a Normal Mouse IgG as a negative antibody control and signal from this antibody can be used to determine background fluorescence. 11
10. Assay Performance and Specificity All the antibodies used in this assay are IP positive and were previously validated by mass spectrometry of the immunoprecipitated protein (data not shown, but available upon request). Assay specificity was demonstrated by using primary fibroblasts cell lines from patients with a well characterized enzymatic deficiency in one of the FAO enzymes. Figure 1 shows results after testing cell lines with well characterized mutations and deficiencies in these enzymes (1) ACADVL:p.[N122D], (2) ACADM:p[K304E and (3) HADHB:p[R61H];[R247H]. Although the mutation was in the HADHB gene, levels of HADHA were found depleted by flow cytometry, immunocytochemisty and western blot perhaps due to post-transcriptional instability of the assembled trifuntional protein (HADHA + HADHB). 12
Mean Fluorescent Intensity 4.0 10 5 3.0 10 5 2.0 10 5 1.0 10 5 Levels of ACADVL in control and deficient cell line 0 Control ACADVL deficient Background Mean Fluorescent Intensity 2.5 10 5 2.0 10 5 1.5 10 5 1.0 10 5 5.0 10 4 Levels of ACADM in control and deficient cell line 0 Control ACADM deficient Background Mean Fluorescent Intensity 8.0 10 4 6.0 10 4 4.0 10 4 2.0 10 4 Levels of HADHA in control and deficient cell line 0 Control LCHAD deficient Background Figure 1. Antibody specificity demonstrated by flow cytometry. Control (blue) and deficient cell lines (red) were targeted with the FAO antibody panel against ACADVL, ACADM and HADHA. Background fluorescence was determined with the supplied negative control (black). After background subtraction, the ACADVL deficient cell line shows a 93% reduction in the level of the ACADVL protein, the ACADM deficient line shows a 57% reduction in the level of the ACADM protein and the LCHAD deficient cell line shows a 50% reduction of in the level of the HADHA protein. Confidence in antibody specificity is critical to Flow data interpretation. Therefore all antibodies in this kit were also tested for specificity by fluorescence immunocytochemistry under the conditions used for the Flow assay. Furthermore levels of the proteins were independently validated by western blot in control and deficient cell lines. The antibody against ACADVL is only ICC and IP positive and therefore the levels of ACADVL in the deficient cell line were validated with Abcam antibody ab54698. Examples demonstrating the immunocytochemical and Western blot specificities of the primary antibodies used in this panel are shown in Figures 2 and 3. 13
Figure 2. Antibody specificity demonstrated by immunocytochemistry. Cells were processes with the Flow cytometry protocol as explained above. Panel A shows control fibroblasts and panel B shows deficient fibroblasts. Left panel shows staining with anti-acadvl ab, center panel with anti- ACADM ab and right panel with anti-hadha ab. 14
Figure 3. Validation of antibodies and cell lines by WB. Western blot was run on a 10-20% gradient acrylamide gel. Samples were loaded at 40ug/lane for anti-acadm and anti-hadha antibodies and at 20ug/lane for the anti-acadvl antibody. Deficient samples used were the same as those used for the flow cytometry assay. 15
11. Troubleshooting Problem Cause Solution Low Signal Signal not correctly compensated Insufficient secondary antibody Lasers not aligned Second antibody is not compatible with primaries Check positive single color control is set up correctly on flow cytometer and gated/compensated correctly to capture all events. Increase concentration of antibody. Run flow check beads and adjust alignment if necessary Use secondary antibody raised against mouse for this kit. 16
Problem Cause Solution High side scatter background Cells lysed Bacterial contamination Samples should be freshly prepared. Do not vortex or shake the sample at any stage. Do not exceed 500 x g for centrifugation. Ensure sample is not contaminated. Low event rate High event rate Low number of cells Cells clumped High number of cells/ml Run 1x10 6 cells/ml. Ensure a single cell suspension. Sieve clumps (30 µl nylon mesh). Dilute between 1x10 5 cells/ml and 1x10 6 cells/ml. 17
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