Here's is how I do it:

1) Collect cells in a centrifuge tube, spin down, and aspirate off the media.

2) Resuspend the Cells in 1-2 mL media and do a cell count.

3) Let's say I want 100 uL of 1000 cells for a 96 well plate. The concentration I need is 10000 cells/mL

4) Lets say I have 2.5e6 cells/mL from the cell count

5) Use the formula: [current concentration]/[desired concentration] = DF where DW is the dilution factor

So, in my example above (2.5e6 cells/mL) / (10000 cells/mL) = 250

My DW is 250

Since I have my cells in 2 mL and final volume of 500 mL will give me the necessary 10000 cells/mL for 100 uL aliquots of 1000 cells for each well. This is a bit much, and can scale down the volume as needed.

For 1 96-well plate, I need 10 mL or 10000 uL. 10000 uL / 250 = 40 uL

Add 40 uL of cell suspension to 9960 uL media, and voila.

Hope this helps

Dear Aroon,

I suppose you have already have your cells detached from culture flasks. After that, you should add complete medium in order to inactivate the trypsin. Then, you centrifuge 1500 rpm 5 min. Wash the pellet with PBS. Wash again and resuspend with PBS. Count. Adjust the volume as you desire by adding more PBS or saline. Hope I have helped.

Yes you centrifuge the cells at a relatively low rpm. Our lab does 800 for 5 min for our breast xenograft implantation. Then aspirate the medium and gently finger flick the tube to disrupt the cell pellets. Then resuspend the pellet with gentle pipetation up in down (be careful not to cause bubbles, especially if using Matrigel). Make sure you account for the cell pellet volume in your resuspension otherwise your implantations will contain less cells and may not be as reproducible with other experiments.

How many cells do you plan to suspend in 100 ul and what injection route will you use?

Hello , thank you all for the kind replies, yes it is an injectable form. I intend to use a million d122 Lewis lung carcinoma to induce subcutaneous tumour nodules in C57bl mice. I understand there would be some loss of cells during this process as such i have a couple more leading questions and hopefully you guys wouldn't mind answering. Thank you!

-How much extra should i inject per mice ?

- What sized needle should i use?

- How long can the cancer cell lines be stored in PBS?

Also when centrifuging does it have to be a cold centrifuge and subsequently prior injection should the cells be kept cold or warm? I am thinking there should be warm? Should i place the pre-injectable of D122 cells in the water bathe before i transport it to my animals to inject into a tumour?

Here's is how I do it:

1) Collect cells in a centrifuge tube, spin down, and aspirate off the media.

2) Resuspend the Cells in 1-2 mL media and do a cell count.

3) Let's say I want 100 uL of 1000 cells for a 96 well plate. The concentration I need is 10000 cells/mL

4) Lets say I have 2.5e6 cells/mL from the cell count

5) Use the formula: [current concentration]/[desired concentration] = DF where DW is the dilution factor

So, in my example above (2.5e6 cells/mL) / (10000 cells/mL) = 250

My DW is 250

Since I have my cells in 2 mL and final volume of 500 mL will give me the necessary 10000 cells/mL for 100 uL aliquots of 1000 cells for each well. This is a bit much, and can scale down the volume as needed.

For 1 96-well plate, I need 10 mL or 10000 uL. 10000 uL / 250 = 40 uL

Add 40 uL of cell suspension to 9960 uL media, and voila.

Hope this helps

Yes Agree. A million cells per 100 ul is fine. You can even go down 10 fold.

Spin, resuspend in PBS or medium. Count viable cells. Mix gently and

Inject 100ul per mouse. Keep cells mixed in the syringe. Obviously, the fewer

Cells per mouse, the slower the tumor will grow.

Thank you charles, but if i could i'd like to ask some continuing questions. I counted 5.4e6/ml cells, i need 10^8 cells in 100ul thus i would need 10^9 cells in 1 ml.When i divided my current concentration/ desired concentration= (5.4e6/10e9)=0.0054 DF

Since i would be injecting 100ul in mice i need to make approximately 3ml of cell suspension.. So when i divided 3000ul/0.0054 DF=555555ul, this doesn't seem to be right...Am i missing something? Hoping to hear from you again. Many thanks.