Reagents

Preparing RPMI Media for Cells

10% RPMI

          to prepare 100ml

(1) obtain filter (0.22 ul), bottle, place lid to filter on table and quickly place bottle cap on it and attach filter to bottle

(2) Add 10 ml of FCS serum

(3) 1 ml Blutamine

(4) 1 ml Antibiotic/Antimicrotic

(5) 0.33 ml of 2 ME

(6) bring up to 100 ml in RPMI

(7) turn filter on

1 % PBS

        to prepare 50 ml

(1) set up filer (use .22 um 150 ml bottle top filter)

(2) add .5 ml FCS

(3) bring up to 50 ml PBS

(4) turn filter on

EGC: To prepare 5 mg/ml stock

(1) transfer 3ml pyrogen free H2O into 15 ml tube

(2) obtain sterile 2 ml vial

(3) We need milligrams. Scale is grams so weigh out 0.005 g

(4) transfer to 1 ml vial with spatula

(5) add 1 ml pyrogen free H2O to the vial with EGC

(6) vortex vial and let sit 5 min as you label 1 ml vials

(7) Transfer everything from the vial to syringe (suck it up with syringe and green needle) and then filter this into 2nd 2ml vial.

q. you want to prepare vials with [100 ug/ml] how much will u add from your stock?

V₁ (5000 ug/ml) = (100 ug/ml)(1ml)

V₁ = 0.02 ml = 20 ul

q. how about if you want to prepare vials with [50 ug/ml] how much will u add from your stock?

V₁ (5000 ug/ml) = (50 ug/ml)(1ml)

V₁ = 0.02 ml = 10 ul

q. You want to set up an experiment where your wells will have a final volume of 0.5 ml and you want a final [EGC]=50. How much should you add from your 100 ug prepared vials?

We you calculated above that for 1 ml you would add 20ul. So for [final] 50 ug/ml you would add 10 ul and since you are using half this volume would add 5ul.  (you would add 10ul if you are preparing a final [EGC] of 100 ug/ml.

V₁ (5000 ug/ml) = (50 ug/ml)(0.5 ml)

V₁ = 0.005 ml = 5 ul

MDP: To prepare a 1 mg/ml stock from a 5 mg/ml stock

• (if we place the 5 mg/ml stock into 5 ml we will have 1mg/ml) so take 6 ml PBS and filter sterile using small syringe (connect filter to syringe then pipette PBS into syringe, then push syringe into tube)

• place 1ml into 5mg stock bottle

• place 4 ml PBS into 15 ml tube

• transfer stock to tube (WS)

• dispense appropriately

You would have 5000 ul to dispense. So could take say 2000 ul for 100 ug/ml vials (this means 2000/100 or 20 vials) and the rest 3000 for 50 ug/ml vials or 3000/50=60 vials)

q. For your experiment you want to have [MDP]=10ug/ml. How much would you add from your 100 ug tube if your volume in your plate well will be 0.5 ml.

If you take 1ul out you have 1 ug/ml. But want 10 so have 10ul/ml. But since you are using 1/2 this volume you would take 5 ul.

V₁ (1000 ug/ml) = (10 ug/ml)(0.5 ml)

V₁ = 0.005 ml = 5 ul

LPS: Stock is at 1mg/ml

q. how much LPS should you add to a 0.5 ml volume to get a [final]=10ng/ml.

V₁ (1000 ug/ml) = (0.01 ug/ml)(0,5 ml)

V₁ = .000005ml or .005 ul  (since you can not measure this, clear you need a different stock lower [ ]

If you take 1 ul of your stock and put it into 1 ml media, have [1ul/1ml]

V₁ (1 ug/ml) = (0.01 ug/ml)(0,5 ml)

V₁ = .005 ml = 5 ul

q. how much LPS should you add to a 1.0 ml volume (in your well) to get a [final]=10ng/ml?

If you take 1 ul out of your 1 mg/ml stock and add it to 1 ml media you will have [LPS]=1ug/ml or 1,000 ng/ml.

V₁ (1000 ng/ml) = (10 ng/ml)(1 ml)

V₁ = 0.01 ml = 10ul.   So to recap, you would take 1 ul out of your stock, add to 1 ml media, then take 10 ul of this and add it to your well.