Elioelio

I am harvesting adherent cells from a 24 well plate - there should be at least 100,000 cells in each well. I use trypsin and then add 1ml media and put into eppendorf tubes.
I then centrifuge for 5000rpm for 5 mins. (this is is a 6cm radius centrifuge). However, I do not see a pellet of cells. I remove the supernatant anyway, as I know where the pellet should be, and then resuspend in 200ul media. This is then added to a 96 well plate for flow cytometry. I can see under the microscope there are cells there. However, flow cytometry shows me that there are usually only 2000-3000 cells.
I'm guessing the other cells didn't pellet (I checked, they are not still adhered to the well).

When I pellet cells in 15ml tubes from T75 flasks for passaging and counting, they pellet fine, and this is at a much lower xg. (1000rpm, 5mins, in a larger centrifuge).
5000rpm is very fast - I know it it probably too fast. So why is it that not all the cells are pelleted?

Any idea on how to redress this?

Thanks for any help or ideas!

I've been carrying out cell proliferation assays for a while now, and my treatments have been reducing proliferation of my cells in 6 assays. However, in the 7th they have had no effect on proliferation. (Its a timed assay, so 4 separate 96 well plates at 4 time points, using WST reagent - similar to an MTT assay).

However, in this assay, I had to use a different FBS in my media that I usually use (at 10%), as the stock I was using ran out. Could this be a viable reason for the treatments not working? If so, what could be the reasons behind this?
I will try another assay but can't get hold of new FBS until next week, and I have people wanting reports of my results!
Thanks for any help

Does anyone have experience in using hiperfect transfection reagent in HT-29 cells?
So for I've used 5, 10, and 25mM siRNA with 3, 4.5 and 11ul Hiperfect for 48hrs, but am not getting any knockdown. I'm seeding on 24 well plates at 180,000 cells/well.
I was going to start again with 35mM and 50mM and 9ul hiperfect at 48h and 72hrs. But if anyone has any tips or ideas that would be great, as it would save me precious time!
Many thanks for any info

I'm almost at my wits end with trying to get my western blots to work...

I keep getting ghost bands (inverse/white bands) where my protein should be. Also a very high background.    11.10.12 Gal3 30s.jpg   8.66K   82 downloads  01010.12 c-jun 40s 2nd.jpg   7.59K   87 downloads

• My proteins are at 25ng
  
• After running the gel I use Thermo scientific Superblock (T20 TBS) blocking buffer for 1- 1.5 hours, 12mls
  
• Add 1:10,000 primary antibody. (I've used c-Jun, Galectin-3, Akt, JNK all from abcam)
  
• 4degC on a rocker overnight
  
• Wash x2 in TBST, then rinse in TBST 3x 5mins (sometimes maybe longer i.e total 30mins)
  
• Add secondary antibody 1:10000 to 12mls TBST, 1 hour
  
• Rinse and wash blot 3x 5mins
  
• I use Supersignal west pico ECL. I blot the blot, then add 3mls on blot for 5 mins
  
• Blot off the ECL, and image 1-40secs

I even stripped one blot yesterday that had ghost bands (cJun), and then added B actin antibody. Nothing came up on the image today  11.10.12 Bactin 5s.jpg   8.63K   66 downloads

Any ideas at all I'd be grateful as we're a bit stumped! Possibly the antibodies are out of date? (2008). Although the B actin antibody should work fine.
Am I washing too much? Too much protein? Too much antibody?
Thanks!