Expression Protocols

Isolation of BMDM from mouse femurs for Affymetrix Array Analysis

Remove femurs (Day 0)

• Use female mice age 8-12 weeks
• Soak extracted femurs in 70% EtOH for no more then 1 minute.
• Remove excess tissue and the "knee" from femurs using a dry Kimwipe, or scalpel.
• Store femurs in RPMI on ice until BM is extracted. Goal is no more than 30 minutes from time mouse dies until BM is extracted.

Flush femurs with complete RPMI

• NOTE: all media/PBS used at every step in this protocol should be pre-warmed to 37oC.
• Media = complete RPMI = 500 mL of RPMI with 50mL of FBS, 5 mL Pen/Strep, 5mL L-glutamine added. Media should be no more then 2 months old.
• Fill a 10 mL syringe with ~ 8.5 mL complete RPMI and affix a 26 5/8 gauge needle.
• Snip off the head of the femur.
• Drill the needle into the "crown" at the other end of the femur (where the knee was).
• Flush 2 femurs using the 8.5 mL complete RPMI into a 50 mL conical tube.
• Disperse marrow using a 10 ml pipet, then strain through a 70 ?m nylon cell strainer into another 50 mL conical tube. Rinse strainer with 5 mL of complete RPMI.
• Count the cells. Use Turk's Solution at 1:1 to lyse the red cells. Count 4 corners. (Turk's Solution = 0.01% crystal violet, 1% glacial acetic acid)
• Add rh-MCSF to cells in complete RPMI just prior to plating (final concentration 50ng/mL). rh-MCSF should NOT be freeze/thawed. Thaw only once and then store at 4oC for up to 2 months.
• Plate 5x106 cells/10 cm dish in 10mL of media on non-TC coated 10 cm Petri dishes.

• Wash - rock plate, aspirate media, add 7-10 mL of warmed plain RPMI (no additions), rock again, aspirate again.
• Add 10 mL complete RPMI with rh-MCSF to each dish.

• Rock plate, Remove RPMI, wash 1X with 7-10mL PBS (warmed) add 7 mL PBS + 1 mM EDTA (PBS/EDTA) and incubate at 37oC for ~ 5 minutes.
• Lift cells off the bottom of the plate using a 10 mL pipet.
• Add cells from 4 dishes to a 50 mL conical containing 10 mL complete RPMI (now has 38 mL in it).
• Remove the remaining cells from the 4 plates by rinsing plates as follows: Add 5mL of PBS/EDTA to plate 1, then transfer this to plate 2, then to plate 3, then to plate 4. Then add this 5mL to the 50mL conical tube. Repeat this process 1 more time (sequentially washing any remaining cells from each plate then adding to the conical).
• Collect cells by spinning at 500Xg for 5 minutes in centrifuge. Aspirate the supernatant.
• You will now have two 50mL conicals with cell pellets. These pellets need to be resuspended and combined. To do this, add 10mL of complete RPMI to the first tube, resuspend the pellet and transfer it to the second tube. Resuspend the pellet from the second tube. Rinse the 1st tube by adding 10mL of complete RPMI to it and transferring to the second tube. Bring the final volume to 40mL by adding an additional 20mL of media.
• Count cells (dilute 1:1 with Trypan Blue solution and count at least 4 corners of the hemocytometer) and replate 1 million cells in 1.5mL of media in a well of a 6-well TC-treated plate in complete RPMI with rh-MCSF.
• Cells should be plated 18-24 hours prior to beginning the experiment.

• We add our ligands/stimuli to cells in wells without changing media.

• Use the Invitrogen protocol for Trizol reagent.
• Remove media and add 1 mL Trizol per well of 6-well plate in fume hood (do not wash cells).
• To check for phenol contamination of RNA measure the absorbances at 260 and 270. The ratio 260/270 should be between 1.1-1.3. A ratio of < 1.1 indicates phenol contamination and the sample should be reprecipitated (we use a sodium acetate-ethanol precipitation with the addition of glycogen as a carrier as outlined in the the Genechip Expression Analysis Technical Manual from Affymetrix). Use the absorbance at 260 to to determine RNA concentration.