Hi all
I had gotten some feedbacks from some of our fellow course-mate that my entries are a little too technical and very hard to understand. So I decided to skip the technical stuffs and go straight in to the topic with simple explanation.
So feel free to ask me any questions regarding any parts that you do not understand in my entry.
Simple 4 steps explanation of how an experiment with the Fluorescent Activated Cell Sorter (FACSAria), also known as a flow cytometers, works:
1) Load the samples onto the FACSAria
2) Data will be acquired
3) Set gating to select out the cells of interest
4) Record the data
Flow cytometry is simply just to analyze the data acquired from cell population that was loaded onto the FACSAria. It analyze by detecting and identifying the cell population of interest that were set aside differently from rest of the cell populations.
Some examples of how this could be done are through the uses of cell markers and certain dyes, like fluorescent-coupled antibodies or fluorescent dyes or fluorescent proteins.
Example of Fluorescent coupled antibodies:
1) Antibodies against CD44 cell marker, coupled with PE fluorochrome
Note: PE stands for phycoerythrin
Example of Fluorescent dyes:
1) Hoechst 33342
Example of Fluorescent proteins:
1) Green Fluorescent Protein (GFP) via cell transfection
Okay, that’s all to my introduction to the flow cytometers and I will stop any further complex explanation here. Hope everyone could have a rough understanding of what flow cytometry is about.
Many thanks
Quan Jun
TG02
Group 08
08 September 2008
Double posting! (1st part: Simple Introduction to Flow Cytometry)
Double posting! (2nd part: SP Profiling in Stem Cells)
Up next is my introduction to my Major Project (MP), which is related to Side Population (SP).
Short Introduction
Side population is a defined population of cells that could be distinctly identified from the rest of the heterogeneous cell population with the use of Hoechst 33342 dye in the flow cytometers. Using defined filters that allow the collection of the emission profile of the used dye does this.
The primary objective of my project is to identify the best blockers blocker and its optimal concentration in blocking ATP-binding cassette (ABC) family of transporters. The blockers are Verapamil, Fumitremorgin C and Reserpine respectively.
We will be using bone marrow cells.
ABC family of transporters is responsible for the high efflux of the Hoechst dye that contributes to the low Hoechst staining which could be identified with the use of flow cytometers. We use isolated bone marrow from murine species for SP identification.
Below is a diagram that shows how SP cells are identified and gated. .JPG)
The following is the protocol in which my laboratory follows to acquire SP profiling in flow cytometry.
(The protocol is only for understanding purposes; so certain information is left out, please understand)
DMEM = Dulbecco’s Modified Eagle’s Medium
HBSS = Hank’s buffer salt solution (Hank’s Balanced Salt Solution)
Use surgical instruments to remove the fur from the fore and hind limbs
Remove and store the fore and hind limbs in cold DMEM+(with EDTA)
Transport the specimens on ice and process in the laminar flow hood
Remove the meat from the fore and hind limbs
Cut the ends of the femur, tibia, humeral and radius
Flush the interior of the bone until it turns white
Filter the cell suspension obtained with a 100um cell strainer
Centrifuge the cell suspension
Remove all but 500ul of the supernatant
Add erythrocyte-lysing solution in a ratio of 11:1
Incubate for 5 minutes under room temperature
Centrifuge the cell suspension
Remove the supernatant and wash 2 times with cold HBSS+
After washing, centrifuge the cell suspension again
Remove the supernatant and add 2ml of cold DMEM+(without EDTA)
Count the number of cells using a hemocytometer
Calculate the total number of cells in the cell suspension
Prepare and label empty 15ml falcon tubes
Pipette in 1mL of pre-warmed DMEM+(without EDTA) in each of the tubes
Calculate the amount of cell suspension to add to achieve a final concentration of 106 cells/ml for each of the tubes
Place the tubes in the waterbath (37oC)
Add blockers to “Hoechst + blocker + PI” tubes according to the blockers to be used
Pre-incubate the cell suspension for 15mins at 37oC
After pre-incubation, add in the Hoechst 33342 dye to all the tubes and incubate for a further 90mins at 37oC
Mix the cell suspension at every 20mins interval during the incubation
After incubation, centrifuge the cell suspension
Remove the supernatant and resuspend in cold HBSS+ with PI
Store on ice and out of light before data acquisition on FACSAria
Record the SP profile acquiredWeek 10 sharings
Hi everyone!
It is time for me to introduce to you the new DropArrayTM platform that my company has developed.
DropArrayTM is a platform aimed to rival the 96-well plate. The 96-well plate is the most common platform used to run assays, especially those that require numerous washing steps. One example of such assays is Enzyme-Linked Immunosorbent Assay (ELISA).
However, running assays on a 96-well plate has been shown to consume relatively large amounts of reagents, samples, and assay time. As for the washing steps, there is the risk of cross-contamination and/or carry-over of liquids across the different wells.
This new platform has been designed and developed to overcome the problems mentioned above.
I will briefly describe the components of the DropArrayTM that I most oftenly use:
S48/S96 Plate
The plates are actually microscope glass slides patterned with Teflon coat. The coat acts as a hydrophobic surface. The S48 plate has 48 wells (12x4), and the S96, 96 wells (12x8). The diameter of the wells is 2mm.
LT100 Wash Station
This station comprises of a chamber and control buttons to set the speed, duration and volume needed for washing. There is an inlet tubing to allow wash buffers to flow into the washing chamber, and an outlet tubing to drain it out of the chamber. The plate is loaded into the chamber and the washing (or rinsing) process can then start: drain any oil > fill chamber with wash buffer > shake > drain wash buffer.
The shaking step is to remove any media or liquid from the wells.
Incubation Oil
For cell seeding. After the cells are seeded into the wells, the wells are covered with incubation oil to prevent evaporation of media during the incubation period inside the CO2 incubator.
Rinsing Oil
Rinsing oil also prevents total evaporation of liquid from the wells when running the assay. What makes the rinsing oil different from the incubation oil is that the former has lower boiling point. So, the incubation oil is more suitable for the CO2 incubator environment. The rinsing oil is also applied to prevent carry-over between wash steps.
There are few other components, such as: Plate Adapter, Dispensing Guide, Tip Cassette, and Lid. But since I seldom/do not use them, I will not explain them here. Feel free to click here if you would like to know more.
The DropArrayTM platform is also used for cell-based assays. I will describe one such assay that I had done in week 9 in the next entry. So, stay tuned!
Nor Liyana
0607927A
TG 02- Group 8