Preparing rotation medium

Stock protocols

The sugar water working solution must be checked for bacterial/fungal growth before cells are passaged!

LPS containing media solution

Steps 1 and 2, as well as 4 through 6 must be done in a safety cabinet (either a fume hood (steps 1 and 2) or a biosafety cabinet (4 to 6)) after proper sterilization of the working area. The working area must be thoroughly sterilized/cleaned after use. Hands must be washed properly after interaction with LPS powder and solution

1.Measure 0,5 mg (i.e. 500 micrograms) of LPS powder and add it to a 15 ml falcon tube.

2. Add 500 microliters of sterile water to the 15 ml falcon tube containing LPS powder.

3. Vortex the tube to ensure proper mixing and dissolution of LPS.

4. Extract the solution from the 15 ml falcon tube with a “insert volume later” syringe.

5. Filter the LPS solution through a “insert filter size” filter into a 1,5 ml Eppendorf tube.

6. Add 50 microliters of the filtered LPS solution into a separate 1,5 ml Eppendorf tube. Repeat this step to make a total of 8 such aliquots.

7. Freeze these aliquots and withdraw as needed when preparing media.

8. Prepare a 50 ml falcon tube with 50 ml heat-inactivated growth media (containing FBS and pen-strap).

9. Carefully add 0,5 microliters of the LPS solution to the media (giving it a concentration of 10 ng/ml).

10. Mark the tube carefully with an ethanol-resistant marker.

Stock sugar water

Done in the v420 lab:

1. Measure 3,33 grams of sugar (farin/sucrose).

2. Fill a 15 ml tube with 10 mL of distilled water.

3. Add sugar to distilled water and shake well.

EDTA working solution protocol

1. Fill a 15 ml tube with 2,25 ml distilled water.

2. Add 250 microliters EDTA from stock solution to the distilled water in the 15 ml tube.

BSA working solution protocol

This preparation should be performed in a clean lab biosafety cabinet:

1. Fill a 15 mL flask with 5 mL distilled water .

2. Slowly add 0,5 g BSA powder to the 5 mL distilled water whilst slowly stirring.

3. Store at 4 degrees celsius.

Kolliphor working solution protocol

Preparation is done in the v420 lab:

1. Add 1 ml of kolliphor to an empty 15ml tube.

2. Slowly add 9 ml of distilled water in small increments to the 15 ml tube containing the kolliphor whilst mixing the two well.

3. Store at 4 degrees celsius.

Protocol dilution sequence

The first 5 steps require use of a bio safety cabinet and must be done in a clean lab. All steps after step 5 can be done in the v420 lab.

1. Remove 0,5 mL of GM with diluted cells.

2. Centrifuge the GM at 400 RpM for 4 minutes.

3. Remove the supernatant without disturbing the pellet.

4. Add 29 microliters of RPMI to the 15 ml tube containing the cell pellet.

5. Add 40 microliters of BSA working solution to the 15 mL tube containing the pellet.

6. Add 1 mL of sugar water mix (33% w/v) (dilute 0,33 g of sugar in 1 mL of water).

7. Quickly add 40 microliters of kolliphor working solution.

8. Quickly add 40 microliters of EDTA working solution.

9. Quickly fill the tube with distilled water until the solution has a total volume of 4 ml, then shake gently.