Macrophage Isolation: When things get cloudy

Macrophage Isolation: When things get cloudy
	Macrophage Isolation: When things get a little cloudy by Heather Buschman
Macrophages are dedicated phagocytes that love nothing more than seeking out infection or inflammation and gobbling up bacteria, dead cells, and other debris. Once activated, these innate immune defenders also release chemokines and cytokines that trigger an inflammatory cascade. In the lab, macrophages are used to test all kinds of things, from phagocytosis to trans-endothelial migration. One of the most common ways to obtain macrophages is to elicit them from the mouse peritoneum. First, healthy mice are injected intraperitoneally (i.p.) with 3 mL of 3-4% thioglycollate. This causes inflammation, triggering monocyte differentiation and localization to the body cavity. Thus, this method results in a higher yield of macrophages than techniques that rely on collecting only resident macrophages. However, thioglycollate-derived macrophages are “activated”, and therefore are not appropriate for every experiment. (Alternatively, murine monocytes can be collected from the bone marrow and differentiated into macrophages, with high yields and little activation.) After 3-5 days, the thioglycollate-injected mice are sacrificed, injected i.p. with 5-10 mL of sterile, ice cold phosphate buffered saline (PBS), and gently massaged. Then the skin is carefully snipped and peeled back to reveal the intact peritoneum, distended with fluid. At this point, a large gauge needle and syringe are used to draw back out as much of the injected PBS as possible, without disturbing the membrane or organs. This extraction should be cloudy with white cells, but mostly free of red blood cells. Keeping this solution cold, the cells are gently centrifuged and rinsed with fresh cold PBS several times before resuspending in tissue culture media. As discussed in the mouse peritoneal macrophages isolation forum at Scientist Solutions®, the easiest method for separating the macrophages from the other white blood cells is by simple adherence. When plated in a tissue culture dish, the macrophages will stick overnight and the rest of the cells can be rinsed away the next day. To increase macrophage yields, several posterssuggest “aging” the thioglycollate. Nobody seems exactly sure why, but everyone agrees that autoclaved thioglycollate stored for a week to several months just works better. Some forum participants also report better results extracting with 16-30% sucrose rather than PBS.

ICMR Junior Research Fellowship -2009

Indian Council of Medical Research (ICMR), Delhi invites applications for its JRF-entrance exam -2009 .


No of fellowship:
120


Subject:
Biomedical sciences with emphasis on Life Sciences (like microbiology, physiology, molecular biology, genetics, human biology, bioinformatics, biotechnology, biochemistry, biophysics, immunology, Pharmacology, zoology, Environment Science, botany, veterinary sciences, bio-informatics etc.).


Fellowship:
Rs. 12000/- pm
To be revised


Qualification:
M.Sc. or equivalent degree with minimum 55% marks for General/OBC Candidates and 50% for the SC/ST & physically handicapped candidates .


Important dates:
Date of examination- 12 July 2009.
Deadline to apply - 29 April 2009


To download application form and more details,
ICMR- JRF exam details
PhD scholarship

USE OF THE LIGHT MICROSCOPE

• Each time the microscope is to be used it should be set up correctly to give a good image. Most often users forget to adjust the iris diaphragm and focus the condenser to give its optimum performance, this could result in objects in the preparation being undetected.
• There are many microscope set up procedures for specific functionality but the most versatile for general use is ‘Critical Illumination’ as detailed below.

1. Examine the microscope and identify the parts as siting of some controls may vary and some have ‘Fixed’ or specialised condensers.
2. Ensure the light intensity control is set to low, turn on the light and increase the brightness to give a white light output.
3. Raise the substage condenser fully and open the iris diaphragm fully.
4. Rotate the nosepiece to the lowest power objective (x4).
5. Rack the stage down using the coarse adjustment control and place the specimen slide on the stage using the retaining mechanism to hold in place.
6. While looking carefully rack the stage up close to the objective lens (do not rack the lens into the slide; this cannot normally occur when a low power objective is selected).
7. Set the eye pieces to the correct width for your eyes so you can look down both together comfortably.
8. Focus the Microscope.
9. Look down the eyepieces with both eyes open and slowly lower the stage to bring the specimen into focus, (use the coarse adjustment control initially and then the fine). Slight movement of the specimen while focusing by using the mechanical stage controls may aid you to find the object. If the image is not sharp focus adjustment of the eyepieces may be necessary. If one eyepiece is fixed and one adjustable, close the eye over the adjustable one. Now focus using the fine control until a sharp image is obtained, (if you cannot achieve this ask for assistance as the problem may lie elsewhere). Now open the other eye (keep both open) and using the adjustment on the eyepiece itself rotate to obtain a sharp image.
10. Focus the Condenser.
11. The condenser should be fully raised to start with. Rotate the nose piece to select the x10 objective and check the focus using fine adjustment. Position the point of a pencil into the middle of the light source over the lamp housing. Using the condenser control, slowly rack it down until the outline image of the pencil becomes sharp and crisp, (this is the ‘Critical’ point for the condenser for optimum performance from where the technique derives it’s name). Note this must be done only after a specimen has been focused on the stage.
12. Set the Iris Diaphragm.
13. When the iris diaphragm is fully open the image is flooded with light and definition is lost due to ‘white-out’. As the diaphragm is closed controlling the amount of light passing through the condenser the image is darkened and outlines appear thickened and defined as more contrast is achieved. Remove one eyepiece and place safely to one side. Close the diaphragm down ,then open slowly until you achieve a dark ring blanking off about 1/5th of the original field of view to give a ‘bicycle tyre’ effect. The correct amount of light should thus be obtained.
14. Examine the specimen, moving it by means of the mechanical stage controls. No further adjustment to the condenser or iris diaphragm is usually necessary to view other slides of the same type/density at low power. The Vernier scale readings may be used to identify and re-locate a point of interest on a slide provided it is a ‘mounted’ preparation and the slide is placed on the stage in the same orientation, i.e. well labeled.