Generation of Publication Resolution Odyssey Image Files

February 21st, 2013
  1. To save a TIFF file at 600 dpi resolution, open the file in Adobe Photoshop.
  2. Select Image Size form the Photoshop Image menu.
  3. Uncheck the Resample Image check box.
  4. Change the Resolution to 600 pixels/inch.
  5. Click OK.
  6. Select Image -> Adjustments -> Levels -> Auto -> OK.
  7. Select Adjustments -> Invert.
  8. label lanes and bands with T tool.
  9. Select Image -> Image size -> 10 inches tall -> OK.
  10. Print Landscape.

I like to save at each step (ex: 600dpi, autolevels, labels).

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Intracellular Cytokine Staining – Methanol Permeabilization

December 27th, 2012

1. Activate cells at 37°C.
2. Spin @ 2200RPM for 3 min.
3. Add 4% PFA for 10 min at room temperature (or add directly 16% PFA in medium for 4% PFA final concentration).
4. Wash 1× PBS.
5. Incubate with ice-cold 100% methanol at 4°C for at least 10 min.
6. Wash twice in TBS + 1% BSA (USE TBS!!! and not PBS and no tween. 1.66mL 30% BSA in 50mL 1x TBS.).
7. Incubate with antibody (1/200) 30min in TBS + 1% BSA.
8. Wash twice in TBS + 1% BSA.

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T-cell Activation

December 27th, 2012

1. Plate T-cells @ 1×10^6 cells/mL, 200uL/well.
2. Activate with 1:200 bead:cell ratio and cytokine milieu in question. If blocking cytokine response, add receptor blocking antibody to cells for 1 hour before adding cytokines and beads. Mix cytokine blocking antibodies with cytokines for 1 hour before adding mixture to cells. This ensures the blocking activity has an opportunity to block the cytokine before it can act on the receptor and signal.
3. Culture cells for 14 days. Check on media pH and replenish at about 7 days.
4. Reactivate T-cells with PMA/Ionomycin @ 0.5ug/mL of each (1:200 and 1:2000 dilution from stock…).
5. 30 minutes-1 hour later add BFA @ 5ug/mL (1:2000 dilution from stock).
6. Culture for 6 hours.
7. If labeling cells CD4+, label for 10 minutes
7. Fix with 4% PFA.
8. Perform ICS from ICS protocol.

Dynal CD3/CD28 Beads-T cell expander
Catalog# 111.31D 2ml
Invitrogen

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Human Myeloid DC Isolation

December 21st, 2012
  1. Wash cells with 1x PBS.
  2. Resuspend cells at 5×10^7 cells/mL in MACS Buffer with 2.5% PHS (15 mL/500 mL PBS).
  3. Transfer 8 mL of cell suspension into a 14 mL round-bottom polystyrene tube.
  4. Add Anti-Human CD32 Blocker 15 uL/mL.
  5. Add the Enrichment Cocktail at 50 uL/mL.
  6. Mix well and incubate at RT for 30 minutes.
  7. Cortex the D Magnetic Particles for 30 seconds.
  8. Add the D Magnetic Particles at 100 uL/mL cells.
  9. Mix well and incubate at RT for 10 minutes.
  10. Bring the cell suspension to a total volume of 10mL ( > 2×10^8 cells) by adding MACS Buffer.
  11. Mix the cells in the tube by gently pipetting up and down 3 times.
  12. Place the tube, without cap, into the magnet.
  13. Set aside for 5 minutes.
  14. Carefully and slowly pipet clear cell suspension from the bottom and transfer into a new 14 mL round-bottom polystyrene tube.
  15. Place the new tube in the magnet for 5 minutes.
  16. Carefully and slowly pipet clear cell suspension from the bottom and transfer into a new 14 mL conical tube.
  17. Spin at 1600 RPM for 5 minutes.
  18. Resuspend in 5 mL of 5% PHS RPMI.
  19. Count cells.
  20. Prepare cell suspension at a concentration of 1×10^6 cells/mL.

MACS Buffer
500 mL 1x PBS
2 mL EDTA
15 mL PHS

5% PHS Media
500 mL RPMI
1.2 mL Gentamycin
5 mL HEPES

Falcon 14 mL Polystyrene Round-Bottom Tubes
BD Biosciences, Catalog # 352057

Silver EasySep Magnet
Stemcell Technologies, Catalog # 19061

Human Myeloid DC Enrichment Kit (Negative Selection)
Catalog # 19061

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Human Naïve CD4+ T cell Isolation

December 21st, 2012
  1. Wash cells with 1x PBS.
  2. Suspend cells at 5×10^7 cells/mL in R.T. MACS Buffer with 2.5% PHS (15 mL/500 mL PBS).
  3. Transfer 8 mL max of cell suspension into a 14 mL round-bottom polystyrene tube.
  4. Add Anti-Human CD45RO 50 uL/mL and incubate at RT for 15 min.
  5. Add the Enrichment Cocktail at 50 uL/mL and incubate at RT for 10 min.
  6. Vigorously pipette Magnetic Nanoparticles and add to cells at 100 uL/mL.
  7. Mix well and incubate at RT for 10 minutes.
  8. Bring the cell suspension to a total volume of 10 mL ( > 2×10^8 cells) by adding MACS  Buffer.
  9. Mix the cells in the tube by gently pipetting up and down 3 times.
  10. Place the tube, without cap, into the magnet.
  11. Set aside for 10 minutes.
  12. Carefully and slowly pipet clear cell suspension from the bottom and transfer into a new 14 mL round-bottom polystyrene tube.
  13. Place the new tube in the magnet for 5 minutes.
  14. Carefully and slowly pipet clear cell suspension from the bottom and transfer into a new 14 mL conical tube if not labeling with CFSE or a 50 mL conical tube if labeling with CFSE.
  15. Suspend in 5 mL of 5% PHS RPMI if not labeling with CFSE or 1x PBS if labeling with CFSE.
  16. Count cells while they spin at 1600 RPM for 5 minutes.
  17. Bring T cells to 10 x10^6 cells/mL 1x PBS at R.T.
  18. Add the 10uM diluted stock solution CFSE (dilute factory vial with 18 uL DMSO to obtain 5mM stock; dilute 1:500 for a 10uM diluted stock) at 1:10 to the T cells (100 uL of diluted stock/1 mL of cell suspension.)
  19. Mix well and incubate 10 minutes at R.T.
  20. Inactivate the CFSE by washing in 5% PHS at 1600 RPM for 5 minutes.
  21. Suspend in 5 mL 5% PHS.
  22. Count cells
  23. Prepare cell suspension at desired cell concentration.

 

MACS Buffer
500 mL 1x PBS
2 mL EDTA
15 mL PHS

5% PHS Media
500 mL RPMI
1.2 mL Gentamycin
5 mL HEPES

Falcon 14 mL Polystyrene Round-Bottom Tubes
BD Biosciences, Catalog # 352057

Silver EasySep Magnet
Stemcell Technologies, Catalog # 19061

CellTrace™ CFSE Cell Proliferation Kit – For Flow Cytometry
Catalog # C34554

Human Naïve CD4+ T cell Enrichment Kit (Negative Selection)
Stemcell Technologies, Catalog # 19155

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