Dear Abdul and Sona, I thought it might be helpful to think through what you need to do tomorrow. 1.

When you see the cells, please briefly note down the colour of the media in each well and also the percentage confluence in each well? 2, Note down if any cells have lifted off during incubation. 3. Very gently wash the cells in plain media (they may well lift off v. easily, if this is the case, don't wash. 4. Add plain medium/trypsin mix as if passaging them, watch under microscope until rounded up and tap 6 well plate gently, taking care not to spill media/cells between wells. I advise you not to do all the plates at the same time as the following steps will take quite a while. Once the cells are suspended in FCB on ice, they should keep fairly stable for c. 1 hour while you do the remaining plates. 5. Individually collect up the well contents into 15ml Falcons, centrifuge, wash once,in plain medium and before centrifuging again, take a very small (e.g. 40 microlitres) aliquot of the cell suspension for counting on trypan blue stain on the haemocytometer ( you should make a record of the number of cells and also the proportion of dead vs alive cells after c. 1 min in trypan blue) I'm afraid this will be rather time consuming, but may well form an important part of your results. 6. After centrifuge, resuspend in ice cold FCB as per protocol and keep on ice, Then continue with the flow cytometry protocol that sona knows all too well! Remember to harvest a couple of extra wells for each of the positive and negative controls. You should be able to get away with 500,000 cells from each tube for flow cytometry. You may well not have time to run the cells on Monday as I suspect that the counting/sample preparation will take a long time. Take great care to apply the correct primary and secondary antibodies- sona will show you where these are abdul. Hope I can help if any questions- do call (I'm down at St Helier). I won't be around on Wednesday, I don't think Iain or Barbara are, either so I doubt there will be a lab. meeting.

Please email me to let me know how tomorrow goes. Please send me a copy of your cell counting results in a table, e.g. with the columns: Sample name (conc'n IL-6, replicate no.), Colour of well mixture Percentage confluence total number of cells in well, number of live cells in well, number of dead cells in well, proportion live/dead cells.

We'll arrange a time later in the week to go through the flow cytometry results when they're available- just let me know when that is. I'm not around on Wednesday, nor on Thursday or Friday afternoons. Best Wishes, Katie

Dear Abdul and Sona, I thought it might be helpful to think through what you need to do tomorrow. 1. When you see the cells, please briefly note down the colour of the media in each well and also the percentage confluence in each well? 2, Note down if any cells have lifted off during incubation. 3. Very gently wash the cells in plain media (they may well lift off v. easily, if this is the case, don't wash. 4. Add plain medium/trypsin mix as if passaging them, watch under microscope until rounded up and tap 6 well plate gently, taking care not to spill media/cells between wells. I advise you not to do all the plates at the same time as the following steps will take quite a while. Once the cells are suspended in FCB on ice, they should keep fairly stable for c. 1 hour while you do the remaining plates. 5. Individually collect up the well contents into 15ml Falcons, centrifuge, wash once,in plain medium and before centrifuging again, take a very small (e.g. 40 microlitres) aliquot of the cell suspension for counting on trypan blue stain on the haemocytometer ( you should make a record of the number of cells and also the proportion of dead vs alive cells after c. 1 min in trypan blue) I'm afraid this will be rather time consuming, but may well form an important part of your results. 6. After centrifuge, resuspend in ice cold FCB as per protocol and keep on ice, Then continue with the flow cytometry protocol that sona knows all too well! Remember to harvest a couple of extra wells for each of the positive and negative controls. You should be able to get away with 500,000 cells from each tube for flow cytometry. You may well not have time to run the cells on Monday as I suspect that the counting/sample preparation will take a long time. Take great care to apply the correct primary and secondary antibodies- sona will show you where these are abdul. Hope I can help if any questions- do call (I'm down at St Helier). I won't be around on Wednesday, I don't think Iain or Barbara are, either so I doubt there will be a lab. meeting.

Please email me to let me know how tomorrow goes. Please send me a copy of your cell counting results in a table, e.g. with the columns: Sample name (conc'n IL-6, replicate no.), Colour of well mixture Percentage confluence total number of cells in well, number of live cells in well, number of dead cells in well, proportion live/dead cells.

We'll arrange a time later in the week to go through the flow cytometry results when they're available- just let me know when that is. I'm not around on Wednesday, nor on Thursday or Friday afternoons. Best Wishes, Katie