I need someone to write a lab report about cell counting. They are not complicated, if you know what you are doing, you will be done in two hours. I will give you the data from the lab. To know more about the report, read the attach file.
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Fluorescence and Cell Count Assays:
Purpose:
• To become familiar with the topic of fluorescence
• To learn how to use the fluorescence spectrophotometer
• To compare two assays used for cell counting
• To process data from a spectrophotometer into a graph to compare fluorescence units to cell count
Background:
It is vital to biology, drug discovery, and medical and biomedical research applications to develop
sensitive methods for quantitating the number of cells in culture. Using a hemocytometer can be quite
accurate, but is tedious and very time consuming for high-throughput screening applications. It also
presents challenges for tissue engineering constructs, such as small diameter vascular grafts, due to
difficulties in removing cells from the constructs for this type of assay. In this lab, we will review two
techniques to determine cell number using fluorescence: Hoechst 32258 dye for a DNA assay and the
AlamarBlue® assay.
Figure 1. Excitation and emission of a molecule.
Source: IHC Staining Methods
Figure 2. Excitation and emission of a cell Source:
http://zeisscampus.magnet.fsu.edu/articles/
basics/fluorescence.html.
Fluorescent materials can absorb light, become
excited, and emit light based on their atomic structure (1). Absorbance, also called optical density, refers
to the quantity of light absorbed by a chemical or biological substance as measured by a
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Spectrophotometer (2). When a molecule absorbs the energy from a photon of a certain wavelength, its
electrons can become excited and move up to higher, less stable energy levels for a short period of time
as shown in Figure 1. Then the electrons lose a small amount of energy as heat and the rest is given off in
the form of a photon (1). The fluorescence emitted by the molecule has a lower energy than the absorbed
light, so the wavelength of emitted light is longer than that of the excitation light (1) as shown in Figures
1 and 2.
A fluorescent compound that emits light upon excitation is a fluorophore. At a certain optimal wavelength
of excitation, called the excitation peak, the fluorophore will emit light at the greatest intensity (1). The
wavelength emitted with the highest intensity is referred to as the emission peak. If any excitation
wavelength, other than the peak excitation wavelength is used, the relative intensity of emitted
fluorescence is reduced (less efficient). A generic excitation and emission diagram is shown above in
Figure 3. The term Stokes shift refers to the difference in wavelengths between the emission and
excitation peaks as shown in Figure 3. Larger Stokes shifts decrease the overlap between the excitation
and emission spectra, allowing for greater detection of fluorescence emission (3).
Many assays require accurate measurement of the concentration of DNA in solution (4). This is often
used for normalization purposes. A common method for determining DNA concentration is UV
absorbance at 260 nm, however, this method has limitations. RNA and proteins can contaminate this
measurement because they absorb at the same wavelength as the DNA, therefore generating
inaccurate results (4).
Figure 3: Generic excitation and
emission diagram with labeled Stokes
Shift and spectral overlap.
Source: http://www.biorad.com/enus/applicationstechnologies/detectionmethods
A variety of reagents such as cellular stains, fluorescent dyes, and fluorophore conjugated antibodies can
be used as probes to detect cellular structures and metabolic activity (5). An important example of a
cellular stain, Hoechst 32258 dye, will be utilized in this lab to quantify the amount of DNA in a
sample. A fluorescence microscope can be used to qualitatively view samples emitting fluorescent light,
whereas a fluorescence spectrophotometer, also called a plate reader, can be used to quantitatively
detect fluorescence in a sample. A plate reader is able to detect absorbance as well as fluorescence. This
lab will require the use of a plate reader to detect fluorescence and therefore determine cell count. The
diagram shown in Figure 4 depicts the light path and basic components of a plate reader.
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Figure 4: Light path and
components of a
fluorescence
spectrophotometer.
Source:
http://5e.plantphys.net/ar
ticle.php?ch=t&id=66
The fluorochrome Hoechst 32258 is frequently used in a sensitive and simple assay to quantify the amount of
DNA in a sample (4). More specifically, the dye functions by binding to the minor groove of DNA with a
preference for AT nucleotide sequences (6). Once bound to DNA, the wavelength of the fluorescence output
of the Hoechst 32258 dye shifts and can be measured with a fluorescence plate reader using an excitation
wavelength of 355 nm and an emission wavelength of 460 nm (4). In this procedure, calf thymus DNA is used
to construct a standard curve from which unknown concentrations of DNA samples can be calculated (4).
Once the DNA concentrations of the samples are determined using the standard curve, those values can be
compared with known amounts of DNA per cell to approximate cell density. In this lab, the conversion factor
we will be using is 6 picograms of DNA per cell.
AlamarBlue® is a dye also commonly used in research labs to determine cell count. This dye contains
resazurin which is blue and non-fluorescent until it enters cells (7). Once inside viable cells, resazurin is
converted into resorufin, which produces bright red fluorescence (7). The scheme of resazurin reducing
to resorufin is shown below in Figure 5.
Figure 5: Conversion of resazurin into resorufin.
Source: http://www.lifetechnologies.com/us/en/home/brands/molecularprobes/key-molecular-probesproducts/alamarblue-rapid-and-accurate-cellhealth-indicator.html#what
After incubation of samples with alamarBlue® dye, the cell count and viability can be determined using
a fluorescence spectrophotometer. The amount of fluorescence detected is proportional to the number of
living cells in the sample (7). It can be used specifically to count viable cells based on metabolic activity.
Damaged and dead cells have lower innate metabolic signal and will therefore generate a proportionally
lower signal than the live, healthy cells in the sample (7).
Materials:
For alamarBlue® dye assay:
• 96-well plate with NIH-3T3 cells already seeded onto it
• alamarBlue® dye
• 2-20 µL pipet (and corresponding sterile tips)
• Gloves
• Aluminum Foil
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• Timer
• Laminar Flow Hood
• Incubator set to 37˚C and 5% CO2
• Fluorescence Spectrophotometer
• Computer with SoftMaxPro software and Excel software
For Hoechst 32258 dye DNA assay:
• Pi Buffer (already prepared)
• Dye Stock Solution: Bisbenzimide (Hoechst 33258) 0.2 mg/mL (already prepared)
• DNA Stock Solution: Calf thymus DNA 100 µg/mL (already prepared)
• 12-well plate with NIH-3T3 cells (previously seeded at unknown densities A, B, and C; n=3 per
condition)
• Cell Scrapers
• Phosphate Buffered Saline (PBS)
• 96-well plate
• Eppendorf tubes and rack
• 15 mL centrifuge tube
• 20-200 µL pipette (with corresponding sterile tips)
• 100-1000 µL pipette (with corresponding sterile tips)
• Pipet filler
• 5 mL serological pipet
• 100 mL glass beaker (for waste liquid)
• Aluminum foil
• Sonicator
• Fluorescence Spectrophotometer
• Computer with SoftMaxPro software and Excel software
Procedure:
Start with the alamarBlue® assay:
- Put on gloves and lab coat retrieve the 96-well plate of fibroblasts from incubator, and gather
other required materials. - Observe cells under microscope and note approximate percentage confluency and morphology
for each condition (Row A, Row B, and Row C). - Bring the plate to the laminar flow hood and add 10 µL of alamarBlue® reagent to all wells with
cells. NOTE: If you do not touch the pipet tip to the liquid within the plate of cells when
depositing the alamar blue, you may use the same tip to transfer alamar blue to all of the
wells. - Gently tap the side of the plate to distribute the dye within the wells.
a. Observe and record the color of the media within each well. - Cover 96-well plate with aluminum foil and place in the incubator set to 37˚C and 5% CO2.
5 | P a g e - Set a timer for 1 hour and move on to DNA assay until the timer goes off.
DNA assay: - Obtain 12-well plate of fibroblasts as well as all other required materials.
- Observe cells under microscope and note approximate percentage confluency and morphology.
- Bring 12-well plate to laminar flow hood and, using the vacuum aspiration system, remove
and discard media from the wells (you may use the same Pasteur pipet for all wells). - Add 1 mL PBS to each well to wash the cells.
- Remove and discard the PBS from wells using the vacuum aspiration system (you may use the
same Pasteur pipet for all wells). - Repeat steps 4 and 5 for a second wash.
- Add 500µL of Pi buffer to each well.
- Using a cell scraper, gently scrape the bottom of the three wells for condition “A” to lift the
cells from the plate.
a. Use the same cell scraper for A1, A2, and A3, and then re-scrape A1 before discarding the
cell scraper. - Transfer the cell solution from the three “A” wells to eppendorf tubes and label them A1, A2, and
A3 respectively. The same pipette tip may be used for all samples in condition A. - Repeat steps 8 and 9 for the remaining conditions using new cell scrapers and pipette tips for
each condition. Label accordingly B1, B2, B3, C1, C2, and C3. - Let the TA know you are ready to sonicate your samples and he/she will lead you to the lab with
the sonicator.
a. Sonicate each tube for approximately 15 seconds. - Return to the lab classroom and prepare working dye and DNA solutions as follows:
a. Dilute the dye stock solution 1:100 with PBS in a 15 mL centrifuge tube (50 µL dye stock +
4.05 mL PBS).
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b. Dilute DNA stock solution 10:1 in Pi buffer in an eppendorf tube (900 µL Pi buffer + 100 µL
of the 100 µg/mL DNA stock). - Prepare the calibration curve in the first two columns of a new a 96-well plate using serial
dilutions as follows:
a. Add 100 µL Pi Buffer to columns 1 and 2, rows A-G.
b. Add 200 µL diluted DNA solution to columns 1 and 2, row H only.
c. Pipette out 100 µL of diluted DNA solution from column 1, row H and add to column 1, row
G. Mix by pipetting the liquid up and down 3 times.
d. Pipette out 100 µL of the solution in column 1 row G and add to column 1, row F. Mix by
pipetting the liquid up and down 3 times.
e. Repeat this procedure for the rest of column 1 up to row B.
f. After mixing the solution in column 1, row B, pipette out 100 µL of the solution and discard
in the waste beaker.
g. Note: No DNA standard will be added to column 1, row A such that the concentration of DNA is
0.
h. Repeat steps 13C-F for column 2.
• Note: See Table 1 for initial volumes for the calibration curves.
• Note: For additional clarification on the serial dilutions for the calibration curves, seek
assistance from the TA’s.
Table 1: Standard Curve plate set up
Initial Pi Initial Concentration of
buffer volume DNA DNA after
Row
(µL) (µL) Dilution (µg/mL)
A 100 0 0
B 100 0 0.15625
C 100 0 0.3125
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D 100 0 0.625
E 100 0 1.25
F 100 0 2.5
G 100 0 5
H 0 200 10 - Add 50 µL Pi buffer to rows A-C, columns 3-8.
- In row A, columns 3 and 4, add 50 µL of sample A1 to the 96-well plate.
- In row A, columns 5 and 6, add 50 µL of sample A2 to the 96-well plate.
- In row A, columns 7 and 8, add 50 µL of sample A3 to the 96-well plate.
- Repeat steps 15-17 for samples B1, B2, B3, C1, C2, and C3 in rows B and C.
- To all wells with sample DNA and calibration curve wells, add 100 µL diluted Dye solution.
- Cover the 96 well plate with foil and let it sit for 10 minutes.
Using the Fluorescence Spectrophotometer: - Alert the TA that you are ready to use the fluorescence spectrophotometer and he/she will lead you
to the lab with the spectrophotometer. - Bring both 96-well plates from the alamarBlue® assay (if the timer has gone off) and DNA assay to
the microscope room where the fluorescence spectrophotometer (plate reader) is housed. - Note any color change observations for each well within the alamarBlue® plate.
- If the SoftMaxPro software and plate reader are not already running, the TA will turn it on and
start the software. - Select the Untitled 1 tab within the software and close the “plate set up helper” pop-up window.
- For the alamarBlue® plate, start by clicking on the Protocols tab.
8 | P a g e - Click on Protocol Manager Protocol library Cell Growth and Viability Alamar Blue Cell
Viability. - On the left side of the screen, click Plate 01.
- On the panel to the right, confirm the following settings:
a. Read Mode: Fluoresence
b. Read Type: End Point
c. Wavelengths:
• Excitation: 555
• Cutoff: 570
• Emission: 585
d. Read from the bottom - Click on Settings Read area select the wells containing your samples (including the calibration wells).
a. Also under settings, click on Plate type and select 96-wells, 96 Well Costar blk/clrbtm.
b. Do not change any other settings and click “OK.”
c. If the tray is not already open, click on the Drawer button on the plate reader to open the tray.
d. Place the plate in the tray with the notched corner of the plate facing away from you to the right.
e. Confirm with the TA that your set-up is correct before moving on. - Click on the Read button to close the tray and start the scan of the plate.
- Once the scan is complete, the tray will open. Remove your plate and set aside.
- Click on the plate icon on the upper left corner of the window, then select Export
Plate1
OK. - Create a new folder on the desktop with yours and your partner’s last names and save the file here
as “alamarBlue assay data.” This will save the data from the scan as a text file.
9 | P a g e - To save the data in a file type that can be reopened with SoftMaxPro, click on the plate icon in the
upper left corner, then select SaveAs and save the file in the same folder as above with the name
“alamarBlue assay SoftMaxPro.” - Click the settings button to set up the following parameters for the DNA assay (Note: this will
overwrite the information gathered from the previous read for the alamarBlue® assay, this is OK):
a. Read Mode: Fluorescence
b. Read Type: End Point
c. Wavelengths:
• Exitation: 355
• Emission: 460
• Click the check box for auto cutoff – it should auto set to 455 nm.
d. Plate type: 96-wells, 96 Well Costar blk/clrbtm
e. Read area: select the wells with samples and standards
f. PMT and Optics: uncheck the box for “read from bottom”
g. Do not change any other settings and click “OK” - Remove the lid from the DNA assay plate and set appropriately in the tray. Click on the Read button
to start the scan of the plate. - Once the scan is complete, export and save the data file (as a text file) and SoftMaxPro files as
above with the names “DNA assay data” and “DNA assay SoftMaxPro” respectively. - Using a flash drive, make sure each member of your group has copies of the data files for
both assays.
Pre-lab questions: - What is the use of aluminum foil in the two labs?
- What are a few assumptions made when counting cells with the Hoechst 32258 dye DNA assay
and the alamarblue® assay? - How much dye solution would you need for 48 wells (8 wells for the standard curve and 40 wells
for samples)?
10 | P a g e - Why does the emission peak always shift to longer wavelengths than the excitation?
- What happens if your readings are below or above the standard curve? What could you do?
Data/Observations:
Data Table:
Record your observations on the provided data table about cell confluence and morphology
noted for both assays, and color changes during the alamarBlue® assay.
For the alamarBlue® and DNA assays, filled in the pre-made data tables with the relevant
information from your excel data and completed calculations.
Table 1
Qualitative Observations
Observations DNA Assay AlamarBlue Assay
Cell
Confluency
Cell
Morphology
Color
Changes
Other
Table 2
Data Collected During AlamarBlue Assay
Condition
Fluorescence Readings Cell Number
1 2 3 Average Standard
Deviation
Average Per
Condition
Average Per
cm2
of Well
Area
A
B
C
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Table 3
Additional Information to Assist with Calculations for AlamarBlue Assay
Slope of Standard Curve Intercept of Standard Curve Surface Area of Each Sample Well (cm2
)
Table 4
DNA Assay Standard Curve Well Data
Row DNA Concentration
(μg/mL)
Standard Fluorescence Readings
Column 1 Column 2 Average Reading Standard Deviation
A
B
C
D
E
F
G
H
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Table 5
Additional Information to Assist with Calculations for DNA Assay
Slope
of Standard
Curve
Intercept of
Standard
Curve
Volume of DNA
Used for
Fluorescence
Readings (mL)
Dilution
Factor
Total Volume of
Sample Within
Each Eppendorf
Tube (mL)
Conversion
Factor
(g of
DNA/cell)
Surface
Area of
Each Sample
(cm2
)
Table 6
DNA Assay Data Condition Sample Number Fluorescence Reading DNA Concentration (μg/mL)
Cell Number First Duplicate Well Second Duplicate Well Average Standard Deviation Total Per Sample Per cm2 of Well Area Average Per cm2 of Well Area Standard Deviation Per cm2 of Well Area
A
B
C
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Lab Report
Results:
Calculations:
AlamarBlue® assay:
- Calculate the averages and standard deviations for the fluorescence readings for each
sample condition. - Using the equation for a line y = mx + b, calculate the average cell number per condition
using the data from your standard curve.
a. NOTE: For your standard curve, the Cell number is set as the x-axis and Relative
Fluorescence Units as the y-axis. Determine the cell number of your sample by using the
slope and intercept from the equation of the line for your standard curve as m and b
respectively and the average fluorescence from your sample as y and would solve for x. - Calculate the average cell number per cm2
for each condition by dividing your answer
from number 3 by the surface area of each well of a 96-well plate.
DNA assay: - Calculate the averages and standard deviations for each sample’s fluorescence readings.
- Using the equation for a line y = mx + b, and the data from your standard curve, calculate
the DNA concentration for each sample. - Calculate the total cell number per sample using the following equation: Calculate cells/cm2
using the following equation:
Calculate the averages and standard deviations for the number of cells per condition and the number of
cells/cm2
per condition (A, B, and C), and compare with a t-test.
Presentation of Results:
- With the standard curve information provided to you, create an x-y scatter plot for the standard
curve: Relative Fluorescence Units vs. Cell number.
a. Use the data from the number of cells per well column for your x-axis and the data from the
average fluorescence readings column for your y-axis.
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b. Include a linear trendline and show the line equation and R2
value on the graph. - Create an x-y scatter plot for the DNA assay standard curve: Relative Fluorescence Units vs.
DNA concentration (µg/mL).
a. Calculate the averages and standard deviations for the standard curve fluorescence readings.
b. Include a linear trendline and show the line equation and R2
value on your graph. - Create a table to include the average cell number per condition and average cell number per
cm2
and standard deviation for each condition for the DNA assay. - Create a table to include the average cell number per condition and average cell number per
cm2
for each condition for the alamarblue® assay.
Post-lab questions: - In three sentences or less, explain which one of these assays you think is more accurate and why
you think that is the case? - List two advantages and two disadvantages to each assay?
- In three to four sentences, explain how these two assays compare with the hemocytometer
and automated cell count methods of determining cell number from the previous lab?
Discussion and Analysis of Results:
Follow the three-part discussion outlined in the lab manual.
References: - Robinson, J. Paul, Jennifer Sturgis, and George L. Kumar. 2009. Immunohistochemical Staining
Methods. Fifth Edition. Chapter 10: Immunofluoresence. Carpinteria: Dako North America. - Olympus Microscopy Resource Center | Confocal Microscopy – Glossary of Terms in Confocal
Microscopy (2012). Retrieved June 23, 2014, from
http://www.olympusmicro.com/primer/techniques/confocal/glossary.html - Detection Methods | Applications & Technologies | Bio-Rad (2014). Retrieved June 23, 2014,
from http://www.bio-rad.com/en-us/applications-technologies/detection-methods - Fluorescent DNA Quantitation Kit Instruction Manual | Bio-Rad. Retrieved June 24, 2014, from
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_9625.pdf - The Molecular Probes Handbook | Life Technologies (2014). Retrieved June 24, 2014, from
http://www.lifetechnologies.com/us/en/home/references/molecular-probes-the-handbook.html - Labarca, C. and Paigen, K., Anal. Biochem., 102, 344-352 (1980).
- AlamarBlue Rapid and Accurate Cell Health Indicator | Life Technologies (2014). Retrieved June 23,
2014, from http://www.lifetechnologies.com/us/en/home/brands/molecular-probes/key-molecularprobesproducts/alamarblue-rapid-and-accurate-cell-health-indicator.html#how