PEC's...I need help.

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angieo2283
angieo2283's picture
PEC's...I need help.

Not sure if this is the correct place to start this topic, but I need some help. I'm doing my research on the induction of macrophage in the peritoneal cavity of mice. I'm doing IP injections on 3 groups of mice. One group will be injected with thioglycollate alone. Group 2 is injected with thioglycollate along with a catalase enzyme and Group 3 is going to be injected with thioglycollate along with a Cholesterol oxidase enzyme. Basically, I'm just going to do cell counts and staining to see which group has the most induction.

I know how to do the injections and I know how to retrieve the cells. My questions are this:

Once I have collected the cells, washed them, and spinned them down, do I need to vortex them to re-suspend the cells to take a sample for slides?

I'm using Wright's stain for staining.

When doing my cell counts with a hemocytometer, do the cells have to be stained first? Or will I be able to distinguish and identify the macrophages? I know the cells will be from the suspension.
Also when doing the cell counts, do I have to do dilutions, or can I just pipette straight from the sample.

I guess these sorta sound like silly questions that I should know the answers to, but I've grown a little rusty on it.

Jason King
Jason King's picture
You shouldn't need to vortex

You shouldn't need to vortex resuspend the cells since you will have spun them down at about 800g (or less). I would resuspend them slowly with a P1000. To be extra careful you could cut about 1mm off of the end of the tip to help prevent shearing (sheering ?).

We use H&E to stain (Hematoxylin and Eosin) and then count using a hemocytometer. You will be able to identify Macs - they could be the largest cells in your sample and the nuclear stain will be distinctive.

Diluting or not is trial and error. I'd dilute a fraction from each test series to count before doing the staining, then you can use the same number of cells from each group in the staining and counting.

Sandy
Sandy's picture
angieo2283 wrote:Not sure if

angieo2283 wrote:

Not sure if this is the correct place to start this topic, but I need some help. I'm doing my research on the induction of macrophage in the peritoneal cavity of mice. I'm doing IP injections on 3 groups of mice. One group will be injected with thioglycollate alone. Group 2 is injected with thioglycollate along with a catalase enzyme and Group 3 is going to be injected with thioglycollate along with a Cholesterol oxidase enzyme. Basically, I'm just going to do cell counts and staining to see which group has the most induction.

I know how to do the injections and I know how to retrieve the cells. My questions are this:

Once I have collected the cells, washed them, and spinned them down, do I need to vortex them to re-suspend the cells to take a sample for slides?

I'm using Wright's stain for staining.

When doing my cell counts with a hemocytometer, do the cells have to be stained first? Or will I be able to distinguish and identify the macrophages? I know the cells will be from the suspension.
Also when doing the cell counts, do I have to do dilutions, or can I just pipette straight from the sample.

I guess these sorta sound like silly questions that I should know the answers to, but I've grown a little rusty on it.

When you follow the correct protocl you will get plenty of macrophages in the peritoneal cavity and all you have to do is to stain them and check the viability with trypan blue you don't need to vortex.
Inject 0.5 ml aged thioglycolate (can be purchased from Sigma) into the peritoneal cavity of the mouse. Mouse will respond by a local inflammatory reaction. After 3 days kill the mouse and inject 3ml cold complete media into the peritoneal cavity, gently massage the abdomen area and remove the media+macrphages using a pasteur pipette. You can repeat this until the liquid is almost clear(all the cells are removed). Centrifuge for 10 minutes in 1200 Rpm and repeat twice. Resuspend cells in 5 ml complete RPMI and count in a heamocytometer.

angieo2283
angieo2283's picture
Thanks! Thats really helpful

Thanks! Thats really helpful, I appreciate it. I have another question. I'm going to induce macrophage also using Catalase Micrococcus lysodeikticus in one group of mice and Cholesterol Oxidase from Pseudomonas fluorescens in another group. My question is this. One came in a liquid form and one came in a powder form. What do I need to do in order to dilute them to where I don't over overdose the mice and kill them. I'm going to inject these with thio, but I havent been able to figure out how much of each I need to use.