Hello Udeshi Wickramarachchi

Some additions/deletions that you could incorporate in the protocol. Otherwise the protocol is okay to follow.

1. Wash the cells with DPBS.

1. Wash the cells with sterile DPBS.

2. Add Trypsin and incubate for about 2mins.

2. Add Trypsin and incubate for about 2mins.

3. Add the media to stop the trypsinization.

3. Add complete media containing FBS to stop the trypsinization.

4. Transfer the whole cell suspension in the dish to a 15mL conical flask with 10mL of fresh media in it.

4. Transfer the whole cell suspension in the dish to a 15mL conical flask with 10mL of fresh media in it.

5. Centrifuge

5. Centrifuge at 1500 rpm for 10 mins.

6. Discard the supernatant.

6. Discard the supernatant.

7. Add media to the conical tube with cell pellet and blow well to mix the cell suspension.

7. Tap the pellet gently and add complete media to the conical tube, pipette the media up and down a few times to mix the cell suspension.

8. Pipette the cells and media to the 6-well plate in the respective volume.

8. Pipette 2-3ml of cell suspension from the tube in each well of the 6-well plate.

Best.

Thank you very much! Malcolm Nobre

Hi

Beside what Malcolm Nobre suggested, you may also want to do a brief count after centrifugation and resuspending your cells depending on at what time you want to isolate the RNA. Too much cells at the time of seeding may lead to overcrowding and eventually they may wither off the surface (I assume you are working with adherent cells) .

Yes, they are adherent cells. Thank you very much Vikrant Mehta

Hi!

If we speak about the passage protocol, yes, that's the way to go, more or less because they are a small diffrences depending on how do you work.

As an example :

2. Add trypsin and incubate for 2 minutes ( well, time depends on how easy is the cell line to de-adhere from the surface + if you put them at 37ºC or if they stay in the cabin.... just experience aquired working with that cell line but as fast as you can is always recommended)

3. Add the media to stop the Trypsinization (other way to do it can be directly take the cells suspended in trypsin out from 100mm dish and put them in 15ml falcon tube with media )

4. Transfer the whole cell suspension in the dish to a 15mL conical flask with 10mL of fresh media in it. ( normally when i use DMEM supplemented with 5 to 10%FBS,by every 1mL of trypsin with suspended cells, i use only 3mL of media.... is cheaper and it works fine).

Summerized, you want to inactivate trypsin with FBS or any other solution that serves to that purpose, and then centrifuge to "clean" the cells and discard the media , then resuspend and pippette -Count them if you are doing anything but a passage-.

Usually i follow Handling information from ATCC.org of the line i´m working with (https://www.atcc.org/products/ccl-221), ask colleages and if i'm transfecting or any other experiment, method section from reliable papers.

Have a marvellous time experimenting with them!

Hi

The ideal cell density for each cell line may differ, and in order to attain the correct confluency, it may be necessary to modify the volume of cell suspension added to each well.

To guarantee that the cells are evenly dispersed across the new culture vessel, transferring cells from a 100mm dish to a 6-well plate needs a few steps.

1 Fill each well of the new 6-well plate with fresh medium. 2 Mark the new 6-well plate's wells with the names of the cells that will be transferred. 3 Aspirate the leftover medium and serum from the 100mm dish, then give the cells a quick wash with 5–10 mL of PBS. 4 When the cells have detached from the dish, add 2-3 mL of trypsin-EDTA solution to the cells and continue to incubate at 37°C. Typically, depending on the cell line, this takes two to five minutes. 5 Add an equal volume of full medium containing serum after the cells have detached to counteract the trypsin.6 To pellet the cells, transfer the cell suspension to a 15 mL conical tube and centrifuge at 1000 rpm for 5 minutes. 7 To attain the required cell density for each well of the 6-well plate, aspirate the supernatant and resuspend the cell pellet in an adequate volume of complete medium (about 1-2 mL). 8 Fill each well of the new 6-well plate with the required amount of cell solution, being sure to distribute the cells equally across their surface. 9 See the cells' growth over time while they are incubated at 37°C with 5% CO2.

To add the cells cultured to a 6 well plate ... after centrifigation and discarding the supernatant, count the cells on a haematocytometer or a cell counter ... so depending on the count you can add the media and distribute the cell suspension in 6 well plate

Hi Shraddha!

This is what I would do:

1. Wash with 2 mL DPBS

2. Add 1 mL Trypsin and incubate for about 2-5mins, or whenever the cells detach.

3.Add 4 mL media to stop the Trypsinization

4.Transfer the to 50 mL conical and measure cell density with either cell counter or hemocytometer.

5. Dilute accordingly and pipette cells to the 6 well plate.

You don't need to spin the cells down to seed a new plate of cells.

i never use DLD1 cell i always use Hela cell and my procedure is always like you just after the centrifugation and discarding the supernatant I always count the cell and the add medium depending on the cell count. then I distribute the cell suspension into my desire well.

Hi Hannah ... thankyou ... I will try this protocol next time for sure ...