I do follow the same protocol with few changes. After isolating PBMCs by lymphoprep, i leave cells in culture dish for overnight (around 10-12 hrs) for surface adherence. And After overnight, i remove all the non-adherent cells, wash the plate with DPBS and further for staining.

In a 6 well plate i seed atleast 3-5 Million PBMCs. If you assume monocytes to be 5-10% than would be having 3-5 Lacs cells for my Flowcytometry.

I do follow the same protocol with few changes. After isolating PBMCs by lymphoprep, i leave cells in culture dish for overnight (around 10-12 hrs) for surface adherence. And After overnight, i remove all the non-adherent cells, wash the plate with DPBS and further for staining.

In a 6 well plate i seed atleast 3-5 Million PBMCs. If you assume monocytes to be 5-10% than would be having 3-5 Lacs cells for my Flowcytometry.

Dear Monara,

I would suggest adjusting your protocol as follows:

1. Transfer the Buffy coat from fresh heparinized blood into PBS 2mM EDTA (1:5 v/v)

2. Centrifuge at 100xg, 10 min RT with brake

3. Discard the supernatant and resuspend the pellet in fresh PBS 2mM EDTA

4. Centrifuge at 70xg, 10 min RT with brake

5. Discard the supernatant and resuspend the pellet in normal PBS

6. Transfer your PBMCs in PBS into the 24w plate and incubate at 37°C for 1 hour.

7. Check your cells under the microscope: You now should have a lot of monocytes and lymphocytes adherend

8. Change PBS to RPMI with 10-20% FCS without washing

9. After approx. 24 hours/next day, you can wash the left over lymphocytes off.

Regarding your questions:

For IF, I use coverslip dishes. I fix the cells with PFA in RT, them, incubate with primary antibodies at 37° in 0,2% X-100 T + 5% BSA. I wash them using a p1000 and PBS. My mounting media is one homemade with glycerol (I put enough to cover almost half of dish height).

---> If approbriate for your experiments, try ibidi µ-slides or other slides. They are perfect for IF and confocal IF microcopy (example: Macrophages phagocyte zymosan-Cy3 targets)

Washes other than specified above: using PBSx1 or washing buffer (contains saponin) - centrifuge for 5min, 1200rpm, 37°C (or 4°C when using saponin).

---> Why do you use saponin in your washing buffer? Do you stain for intracellular antigens?

Best regards, Johannes

Amalia Naranjo Lucena thank you, Yes I know, it's bizarre. We did not change the dishes material/lot. It's the same company. About solutions, we made new ones. I guess the problem is many washes during the protocol. I though too much mounting medium could also detach my cells, but I use less than Johannes Zeller and for him, it seems not been a problem. Then, I have tried adhering monocytes in coverslip chambered glass and worked better. However, as more cells attach on this glass, more non-specific cells (some lymphocytes and others not identified) attach as well.

Yes Jitendra Kumar Shandilya , I forgot to say that, but I tried putting overnight for adherence and it was worse.

Thank you a lot Johannes Zeller , I'll try your protocol too. 1h in PBS before RPMI could attach better monocytes and make difference.

Thank you guys for your inputs!

Monara

You are most welcome - let me know if you have further questions! Best of luck to you!

Dear Monara,

Monocytes can be further separated from lymphocytes using density gradient centrifugation because they are less dense and slightly larger. In other words, monocytes are faster to float during centrifugation in a density gradient than lymphocytes.

problems are there

1. To maintain density gradient uniformly

2.Flask/plate to adhere the monocytes

3.Faster float of monocytes, controlling is a problem.

4.Tube must be checked before use that is a problem

5. Exact time hours how much?

Ashish

Thank you Ashish Thakur for your input.

Do you know the best rpm/min for centrifugation in order to have more monocytes than lymphocytes adhered on plate?

Again, thanks.

Monara

Dear Monara Kaélle Angelimm,

RPM depends on the density/concentration of cells per plate. Various suitable conditions can be applied here before using RPM values according to nature of isolation.

In general sense, human blood should put the cells in culture with PHA for 3 days prior(blood diluted 3-fold), centrifuged at 18-20°C for 30 min and to add 20 mL of buffer at 400-600 Xg and centrifuge 1200 rpm-1600 rpm, 10 min to wash is most beneficial for finding results. Dendritic cells may be generated from monocytes by culture; Plating more densely than ~1 × 108 cells per plate reduces adherence and final in such case centrifuge the gradient 10 min at 800 × g (1800 rpm in H1000B rotor(capacity 4 x 250 mL) to resist corrosion on plate) may be allowed. In case of a monocytes/macrophage-like trout cell line, centrifuging at 1000 rpm just for 5 min in a table top centrifuge is sufficient. In case of fetal calf serum (FCS), centrifuge 10 minute at 1300 rpm (400g, 18°C) is sufficient to get quite good results. For lymphocytes isolation can be centrifuge at high rpm more than 2000 rpm at 10 minute because the high concentration of cells per well during the interaction of monocyte- lymphocyte 2X108 cells. If concentration is more you need to use more RPM for isolation. Tissue culture flasks, dishes and plates of various sizes, can be used in an effort to establish more of the cells adhered.

Ashish