hey,

I think the cells which are adherent look great.

the reason why your cells swim around after some time might be because they die.

Do you use enough FCS?

Do you use also M-CSF?

I never had such troubles but I think it might be a medium (I used DMEM with high glucose) issue.

best,

Sebastian

thank you Sebastian Wienerroither for your answer. I dont use M-CSF,

here is my brief protocol.

Sacrifice mice. Cut peritoneal cavity and pull back skin. Pull membrane away from mouse body and carefully insert needle and inject 5ml 1640 (GIBCO RMPI 1640,cat: 31800-022, we filter the medium in our lab, however, there do been some dust because of the container, that maybe a concern) into peritoneal cavity.

shake the entire body several times, and collect peritoneal fluid with 5ml pipette.

Centrifuge at 1500 RPM, 5 min at 4℃.

Pour off sup.(without washing)

Resuspend cells in 1640 + 10% FBS + 1x Pen/Strep + glutamine. Seed one well of 12-well plate for one 6~8w balb/c mouse.

Let sit for 1 hrs, wash 2x to remove non-adherents

Incubate overnight and then use.

Are there any issues with this protocols?

Why is incubate overnight?

The incubator Males Sense because the pems Might be stressed.

I think the Isolation should be fine.

I centrifuge the cells at 25C.

I think you should change the Medium a Bit. Add also MCSF and also fcs.

This should help.

Maybe you have also Too many cells on One plate. I counted the cells and used 2000000 cells per 3,5cm dish.

Best

Sebastian

Hi Lei,

Medium may be a issue, but unlikely. Also look at whether you are using the right culture plate or not. I think your problem may be overcrowding and hence using up resources. Also, if a second layer of cells attach to you first layer, it can pull off your initially attached cells, leaving gaps (I do not see it on your photo though). What is the recommended seeding rate for your macrophages? Even then you would need to optimise the number of cells you are seeding per cm square area. You mentioned that you seed one well of 12well plate from one mouse. You would need to count how many cells you get and then seed the right amount. Make sure you record the number of live/dead cells. I guess for macrophages you would need viability of over 80% (check in literature). If you have too many dead cells, their products are toxic to the healthy cells. Is it possible to add a washing step to remove dead cells? Also, in general with many cell types it is well accepted to allow cells to attach longer (at least 2hr, preferably 4hrs) before washing and incubating further. This allows adequate attachment. Also, do you use the same amount of FCS in the last incubation step. At this point high FCS can be more damaging than useful.

the media is okay. maybe check on the amount of medi u using. maybe u are using a small media with a large number of macrophages. I think the floating of macrophages means they are dead because live macrophages adhere to the surface. macrophages are incubated for 4 hrs at 5% CO2 and at 37 o C. the conditions should mimic the mice or human body. check well you may find that one of these conditions are missing. thanks

Confluency of cells during adherance really matters.if u try seed more cells than usual and look after overnight incubation....

hi Lei

Detachment of cells always caused by the following:

1- The type of the attachment factor of your culture plate

2- The type of media you have used

3- PH of the medium

4- C02 percent

5- Media additives

6- Density of cells

So, you have to check the above mentioned conditions.

Also, i would like to tell you that i isolated the same cells by the same method but with some differences:

1- I injected the cavity by cold phosphate buffer with 3% FCS and collected the cells by both syringe and sterile dropper.

2- I used DMEM with 10% newborn calf serum

3- The media additives were 0.1% antibiotic antimycotic, 1% Essential and non essential amino acids. In addition to the 10% serum before hand.

4- I set the Co2 incubator at 5.5 % .

Good luck

Hi Lei,

First of all your adherent macrophages look good enough. I beleive that the problem with floating cells is not the protocol. So, macrophages are differentiated monocytes upon stimulation of varius cytokines of myeloid lineage such as M-CSF. In my oppinion, since you do not separate every cell population by centrifigation with ficol it is possible enough to have more monocytes than actual macrophages. Mind that monocytes grow in suspension.. Try to

1. use MCSF in your culture media, to decrease the volume of floating monocytes, and increase macrophages.

2. decrease the cell density a little because o/n incubation is needed for the recovery of primary cells

3. use a substrate, propably coat your dishes with polylysine prior use in order to facilitate adhesion.

Good luck!!!!!!!!!!!!

I think you are using wrong medium use DMEM instead of RPMI1640

Are these cells coming from a naive mouse or thioglycolate treated mouse? If it came from a naive mouse, it is normal.

I have experienced with RPMI1640 and I have good results. I think you should use cooled medium when washing for removing no adherent cells. Perhaps, you have lymphocytes contaminating your culture.

Hey Lei,

First of all check the media which you have already checked RPMI is good for the mac culture, then check the CO2 incubation chamber the water level and co2 % and most of all dont wait for overnight mac adheres within 1-2 hrs and it is good if you change the media after 1hrs because in the time course thereis compeition for the cells to settle and you wont be able get dense monolayer.

I hope this will help you out..

Hi Lei,

I used to have this problem earlier days of my research. I suggest you to use 0.05 million cells in 6-12 well plate in complete medium containing all your ingredients and 10% FBS and also do not forget to add BME.

Cells should be OK. if you have any questions please let me know.

Bets of luck

Thanks for all your guys’ suggestion.

I don’t know if the cell density is really matters. Since the cells had been washed twice the first day, and attached cellｓ were not very crowded. The problem is why the cells detached from plate after overnight culture, and the next few hours after the medium was changed.

However, there are other classmates in our lab, they say they don’t have this issue, and I will watch how they handle their cells, and will let you know the result.

Hi Lei,

Cell density matters for consistency (you want to be able to reproduce your conditions), especially if you want to publish. The percentage of viability also matters, as if you have too many dead cells after seeding, the breakdown products are toxic to your healthy cells. So, even if you have a good surface coverage with healthy looking cells, the healthy cells may be "poisoned" during the first few hours after seeding. Also, if there are too many dead cells (for a lot of cell types 80% and over viability is acceptable), the QUALITY of your cells in general may be reduced. If so, then although they initially look healthy, they may not be able to stay healthy and then die overnight and float off. Although most of the time cells round up and float off, some cells can stay attached even if dead. The breakdown products from these cells then poison other attached cells, making them to detach. Have you checked the viability of your cells on the plate? Although I do ot have much experience with your cell type, once cells are processed, some are bound to die during the process.

Cheers

Try to handle your cell gently and try to digest it with Trypsin shortly when you try to passage them.Do leave in 37 degree incubator when you digest it with trypsin. Good luck.

I have experienced similar problem in peritoneal macrophages culture. Please add 10ng/ml M-CSF in your culture medium. That will resolve your problem. Good luck!

I would not use trypsin for macrophages.

It will not work that well.

you could use citrate buffer or a mixture of EDTA and PBS (but I always used a soft scraper).

and yes M-CSF is a good idea!!

It is important to handle primary cells very gently. Otherwise, they may look healthy at first, but many will die overnight. All techniques, from harvesting to seeding in the plates must be gentle on the cells. Don't centrifuge at too high of a speed. After washing, decant the supernatant, then gently swirl the cells in the residual liquid until homogeneous. Don't resuspend by rapid pipeting up and down or banging the tube on an object. If using pipet tips to seed in wells, use wide bore pipet tips.