Please explain what the experiment is showing me. Discussion answer 2-3 paragrap
ID: 264056 • Letter: P
Question
Please explain what the experiment is showing me. Discussion answer 2-3 paragraphs long.
IL-1? Enzyme Linked Immunosorbent Assay (ELISA):
Samples: supernatant collected from LPS stimulated macrophage cell culture. Macrophages are seeded onto wells and 50 ng/ml, 25 ng/ml, and 12.5 ng/ml of LPS doses were added to the wells for a 4 hour incubation. After 4 hours, supernatants were collected and were frozen down at -70°C for the ELISA assay in class. Our hypothesis is that if LPS activated macrophages release the active form of IL-1?, we would be able to measure a dose dependent level of IL-1?.Our control groups would be macrophages without LPS, which would show us that LPS is the only component that induces cytokine induction.
Material: 96-well plates , Wash buffer in a squirt bottle, 1000p or 200p with tips, Trash beakers or paper towels for waste tips, Secondary Antibody conjugated with Biotin (3.5ml), Streptavidin-Horseradish peroxidase conjugate (HRP) (1.7ml), Substrate (TMB/), Stop solution, Standard (2000pg/ml Il-1?) and 7 empty tubes, Zero well sample buffer, 3 samples: Supernatants of untreated and LPS (50 & 25 ng/ml LPS) treated macrophages.
Methods:
First make a series of standard samples: These are known concentrations of samples that will be used to make a standard curve to extrapolate unknown sample results quantitatively. 150ul of 2000pg/ml IL-1? will be given to you. Carry out 2 fold serial dilutions of 7 tubes using sample buffer as a diluent 1000pg/ml, 500pg/ml, 250pg/ml, and on…
1. Add 100ul of samples and standards onto designated wells.
Standards into each well
Leave 2 wells empty. These are your blank wells to blank spectrophotometer
Samples 1, 2, & 3 into remaining wells in duplicates
2. Incubate for 20-30 min at room temp
3. Aspirate out and wash all wells with wash buffer (3X wash)
Discard liquid by inverting the wells onto stack of paper towels
Add wash buffer (fill it up) and discard as before. Repeat washes 2 more times
4. Add 100ul of secondary Ab conj. With Biotin into each well except blank wells
5. Incubate for 20-30min at room temp
6. Wash 3X as before
7. Add 100ul of Streptavidin-HRP conjugate and incubate for 10-15min at room temp
8. Wash 3X
7. Add 100ul of substrate into each well including blank wells
8. Incubate for 10 min under dark (cover the plate completely with Aluminum foil or paper towels)
If the color is not blue, leave for 5 more min
9. Add 50ul of stop solution (2N Sulfuric acid). No bubbles.
10. Get OD reading at 450nm
Blank the spect. with blank wells.
11. Analyze the data
The result section of your lab note should include both a standard curve as well as sample results (A table is fine).
Explain what those numbers mean to you under the discussion section.
Explanation / Answer
Please explain what the experiment is showing me. Discussion answer 2-3 paragrap