MTT assay

Hello, i am Stanley and this is my second blog posting.

The MTT assay is a quantitative colorimetric method used to determine cell proliferation. It utilizes the yellow tetrazolium salt which is metabolized by mitochondrial succinic dehydrognase activity of proliferating cells to yield a purple formazan product. This purple formazan is then solubilized and the concentration is determined by reading under an optical desity which ranges from 500nm to 600nm. As the MTT assay only detects living cells, it can be use to measure cell cytotoxicity, proliferation or even activation.

Part of my experiment will include the use of MTT assay to determine both the growth curve of both normal lung cell (MRC-5) and large lung carcinoma cells (H460) which are seeded in a 96 well flat bottom plate. The advantages of using MTT will include high accuracy and the exclusion of using radioisotope.

Below are steps for performing the MTT proliferation assay:

Preparation of MTT stock

1) Adding 1ml of sterile PBS into the 5mg vial of MTT

2) Votex the mixture till all of the MTT is dissolved (takes approximately 3 to 5mins to dissolve completely)

(Note: the MTT stock can be stored for four weeks at 4 degrees celsius protected from light)

Labeling of cells


(Before adding the MTT stock)


3) Add 10µL of MTT stock to each well ( The wells will turn to a slight yellow in colour)

4) Incubate at 37 Degrees Celsius and 5% carbon dioxide concentration for 4 hours (1st incubtion)

5) After incubating the plates for 4 hours, remove all but 25 µL medium from each well

6) Add 50µL of DMSO into each plates

7) Incubate the plate for the second time at 37 Degrees Celsius and 5% carbon dioxide concentration for 10mins (2nd incubation)


(After the second incubation)


Absorbance reading

8) Read the absorbance at 540nm using a microplate absorbance reader

The above steps is a quick alternative to the conventional steps which uses SDS sodium dodecyl sulfate - HCL solution instead of DMSO to solubilize or dissolve the purple formazan. However, the conventional steps will require an addition of 4 to 18 hours of incubation time ontop of the first incubation stated in step 4 and has to be read at 570nm instead of 540nm.

Some important points to note are that:

1) DMSO is a powerful solvent and is able to rapidly penetrate the skin and it is important to avoid skin contact with DMSO

2) Both DMSO and the MTT stock are sensitive to light and must be kept away from light to prevent degenerative effects or inaccuracy

Thank You

slide making

hihi, im going to share about slide making of blood specimen for chromosome study.

when the blood culture is ready for harvesting...

1. centrifuge the tube of cell suspension at 1200rpm for 10minutes

2. remove the supernatant and resuspend the cell pellet with fresh fixative
[do not cause too much mechanical disruption to the cells]

3. centrifuge the tube at 1200rpm for 10minutes

4. place the tubes ready for slide making in a rack and prepare a clean recycle tube with pipette for each tube in a rack

5. remove the supernatant into the recycle tube, leaving a suitable amount of fixative to resuspend the cell pellet

6. take a slide from a beaker in the fridge and flick to remove excess water leaving an evenly thin coat of water on the surface of the slide.

7. hold the pipette approximately 1-2 inches above the slides
[tilt the slides at about 45 degree angle]

8. gently drop the cells suspension on the slide

9. dry the sides and edge of the slide and leave it at room temperature to dry

10. check the mitotic index and spread length under the microscope

11. bake the slides at 90 degree celsius for approximately 1 hour 30 minutes


Factors affecting slide making

a) Proportion of fixative
if the metaphases does not spread well, use a higher proportion of acetice acid to methanol. this will soften the cell "sac", thus chromosome spread is better. however, this must be handled carefully as increase amount of acetic acid can cause staining problem later on.

b) Cell density
if the cell density is low, make a denser cell suspension by resuspending the cells with a lesser volume of fixative and vice versa.

c) Temperature of slides
warm slides allow more spreading, thus use it when the spreading is tight. cold slides prevent over spreading of the chromosome. however, cold but dry slides is used to prevent further spreading of the chromosomes. it is normally use when cells are "severely broken".

d) Aging of slides
slides can be sged at 90 degree celsius for 1hour 30minutes or 60-65 degree celsius ovenight.

e) Angle of slides
the angle of the slides is adjusted according to the condition of the metaphase. for example, a steeper angle will create a greater flow direction, thus enhanced the spreading.

by,
yeo sok kian jocelyn
0703359j

Staining

This method of staining is called immunostaining. It makes use of antibodies, primary and secondary.

This is a rather simple but tedious task. The steps are:

1. Fix cells using 100% ethanol. (This is to prevent the cells from washing off, during the wash step)

2. Incubate for ~20 minutes in the freezer.

3. Remove ethanol and wash thrice using PBS.

4. Add 4% blocking solution and incubate for 1hour on rotator at room temperature. (Blocking solution used here is just BSA, bovine serum albumin)

5. Wash thrice using PBS to wash off any unbound BSA.

6. After 1 hour, add primary antibody (Dilution ratio--> Ab: BSA = 1:100) and incubate on rotator for 1hour at room temperature/incubate overnight in freezer.

7. Wash thrice with PBS to wash off any unbound primary Ab.

8. Add secondary Ab (Dilution ratio--> Ab:PBS = 1:100) and incubate for 1 hour on rotator at room temperature.

9. Wash thrice with PBS.

10. Add DAPI and incubate for 5mins on rotator at room temperature.

11. Wash thrice and proceed to view under fluorescent microscope.

Note: DAPI stains for DNA(Nucleus). It will appear blue under microscope.

The antibodies that i use are:

1. Rabbit anti-ZO-1 (Primary Ab)

2. Alexa Fluor 594 goat anti-rabbit IgG (Secondary Ab)

Rabbit anti-ZO-1 will bind to the ZO-1 protein. This ZO-1 protein can be found only when the cells are in contact with each other.

Alexa Fluor 594 goat anti-rabbit IgG is derived from goat and binds specifically to rabbit Ab. Therefore, when the above two Ab are used together, Alexa Fluor 594 goat anti-rabbit IgG will binds to rabbit anti-ZO-1. When view under microscope, the protein will appear red.

Below are some of the photos i taken using the microscope:

Note: Permission were taken from my mentor before the posting of these images, except for the last image. The last image was taken from website: http://www.biotechniques.com/BiotechniquesJournal/2007/August/Murine-nasal-septa-for-respiratory-epithelial-air-liquid-interface-cultures/biotechniques-43042.html?pageNum=4

The yellow box shows the ZO-1 protein that I'm talking about. The reddish thing is the cytoplasm, which is overstained. But for the image below, the distinct red lines are the ZO-1 proteins. The green stain is type IV beta-tubulin stain, according to the website.

Reference:

BioTechniques, 2009. Retrieved on 27 August 2009 from website: http://www.biotechniques.com/BiotechniquesJournal/2007/August/Murine-nasal-septa-for-respiratory-epithelial-air-liquid-interface-cultures/biotechniques-43042.html?pageNum=4

Alvin

Hematology (:

Hey people! This is Siew Ming here to post again (:
I am going to post about my experience when I was attached to the Hematology lab.

Basically, the hematology lab is being divided into two sections: 1) Stat lab where emergency samples were being handled and 2) Routine lab whereby routine processing of blood samples are carried out.

I got the chance to observe and participate in the various tests that are carried out in the lab, such as FBC, ESR, reticulocyte Test, Kleihaur Batek Test and APPT/PT. Besides these, I felt fortunate to be taught to recognize and differentiate the different types of blood cells under the microscope by a very senior med tech! (:

Today, I’m going to share about the techniques of Erythrocyte Sedimentation Rate (ESR). ESR is a screening test used to detect inflammation. However, confirmatory tests have to be carried out to confirm the diagnosis. ESR refers to the rate at which the red cells sediment over time. It is often measured in mm/H. ESR will increase when there is infection, pregnancy, SLE, Anaemia, etc.

For a single ESR test, a minimum of 350ul of venous blood will be collected in an EDTA tube. Before performing the test, it is very crucial to check for visible clots of the blood. This is because clotted samples cannot be processed and the results will be inaccurate and not valid.

Steps:
1. EDTA blood sample is mixed well by inverting the tube several times.
2. 320ul of blood from the EDTA tube is transferred into a Aquisel Tube (filled with Trisodic Citrate 0.106M)
3. The Aquisel tube is then mixed for at least 12 times.
4. A pipette is then introduced into the Aquisel tube through a twisting the pipette in a circular motion, and gradually push the pipette downwards to allow blood to fill the pipette. The blood level should reach the “0” marking on the pipette.
5. The pipette, together with the Aquisel tube is left to stand on a Styrofoam Aquisel Tube holder for 50 mins.
6. Results (no. of mm the cells have fallen) were read at the end of 50min.
7. Results were then recorded into the ESR record book, patient’s request form and LIS verification system.


The expected ranges should be:
Neonates (1-2days): 0-4
Neonates (3 days-1 month), children (2 months – 12 years), and adults (more than12 years): 0-10

I think there is something wrong with blogger these few days. As it doesn’t allow me to post pictures, here’s a link to show how do Aquisel tubes and pipette look like: http://www.swissvacuum.com/products/selecta/Biology_and_histology_equipment/DivisionAnalytique.pdf