Mi objetive is to sort MDM infected from uninfected. MN were purify from PBMC using Miltenyi sytem (CD14). MDM are obtained after 7 days of monocytes culture in low adherence
culture plate. MDM are infected 1h with M.tuberculosis For to remover the infected MDM cool PBS is added an for to remover MDM we make up and down with micropipet. We recovered a little amount of cells.
For example I started an experimento with 20 MN millions and I recovered 5 MDM infected millions¡¡¡¡¡
Someone can help me?
First question, do you have wash steps in this protocol?
Second, do you see a confluent cell layer before infection and after infection?
It's usually the wash steps and with low adherent plates, the cells are lightly adhered that you need to be VERY careful when you wash the cells. Always introduce the medium or PBS SLOWLY down the side of the well, and if you keep the plate tilted, the liquid should lightly go overthe cell layer, and you can pipette and discard immediately.
Hope this helps.
Thanks very much for your suggestions,
In this protocolo we do not include wash steps and after the infecton is very hard to remover the MDM. We use the ultra los adherente culture plates. For to remover MDM we use cool PBS. So after this experiences we are thinking to use trypsine.
Greetings
Yeah, if the low adherence plates are causing the cells to stick that you can't wash off, you will have to try trypsin/EDTA.
I know you mentioned that you pipette up and down in the well with the PBS, I want to add that you should do this with the 1000uL pipette and aim the stream of PBS directly on the adhered cells to flush them off the plate. If they are only lightly adhered, they should come off.
Dear Shen,
I did your suggestions with 1000l pippet and I need to repite this process at least 3 times. I thougth to try with trypsin/EDTA. Do you have any protocol? Now I get the Gibo information and I will do the proceso the next week because now MN are in incubation for to get MDM.
Greetings an thanks a lot
The pipetting is quite tedious. Normally, I would need to pipette up and down at least 10 times, aiming the stream of PBS at various parts of the well, to get most of the cells off the plate. If you are doing a lot of wells, it will be VERY tiring, and your thumb will hurt.
As for the trypsin/EDTA... if you bought the ready to use version, I usually just put enough to cover the cells... So, for a 24 well plate, it's about 200uL/well, and you can scale up accordingly for 12 and 6 well plates. Once you add the trypsin/EDTA, put it in the incubator for about 5 to 10 minutes, depending on how stuck those cell are. You can take the plate and swishing around, and look at it under the microscope to see if the cell have detached. After the incubation, add back 2x the volume of media with FBS, this will help to "stop" the process and give you more volume to work with during isolation
If after 10 minutes, you still see cells attached, once you add the media, they should be released enough that if you do the pipetting technique to get them off.
Hope this helps.
María, it may help if you place the plates on a bed of crushed ice for a few minutes before you attempt to remove the cells. Perhaps they will release better if they & the PBS are kept at 0°C while pipetting.
Dear Shen,
I am goin to follow your recomendations using trypsin and let to get good results.
Thanks
Thanks Leonard,
I previously did your suggestions and is very hard to remover MDM
Geranios
Dear Shen-An,
I am very happy because today I used Trypsin/EDTA for to remover MDM followed your suggestions and it works¡¡¡¡ MDM calles were removed and the cells viability was close to 100%.
Next wednesday I going to infec MDM and I will use Trypsin/EDTA for to remover MDM infected.
Thanks a lot
Geetings from Mexico City
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