IN THIS EXPERIMENT, WE'LL DEMONSTRATE A BASIC PROTOCOL FOR DILUTION CLONING OF AN ATTACHED
CONTINUOUS CELL LINE IN A 6-WELL CULTURE PLATE.
DILUTION CLONING IS THE TECHNIQUE USED FOR CREATING A POPULATION OF CELLS DERIVING FROM
A SINGLE CELL, RESULTING IN A CELL STRAIN.
BY DILUTING CELLS BELOW A CERTAIN DENSITY, INDIVIDUAL CELLS WILL FORM DISCRETE COLONIES.
WE'LL BE USING NIH3T3 CELLS, WHICH ARE COMMONLY USED IN RESEARCH LABORATORIES.
TO BEGIN, REMOVE THE CULTURED CELLS FROM THE INCUBATOR.
VISUALLY INSPECT THE CELLS TO SEE IF THERE IS ANY CHANGE IN PH, WHICH CAN BE DETECTED
BY A DISCOLORATION OF THE INDICATOR PHENOL RED.
EXAMINE THE CELL CULTURE UNDER AN INVERTED MICROSCOPE.
LOOK FOR ANY SIGNS OF CONTAMINATION OR UNHEALTHY CELLS.
THESE CELLS LOOK HEALTHY.
NEXT, CALCULATE THE PERCENT CONFLUENCY.
CONFLUENCY IS THE PERCENTAGE OF THE VESSEL COVERED BY CELLS.
OUR CELLS ARE AT ABOUT 90-PERCENT CONFLUENCY.
TO BEGIN, TRYPSINIZE THE CELLS TO GENERATE A SINGLE-CELL SUSPENSION AND COUNT THE CELL
SUSPENSION.
CLICK THE LINK ON YOUR SCREEN TO WATCH A DEMONSTRATION OF THESE STEPS.
TO PERFORM A SERIAL DILUTION, WE NEED 5 LABELED TUBES.
OUR FIRST TUBE CONTAINS A CONCENTRATION OF 10,000 CELLS PER MILLILITER.
ADD 4.5 MILLILITERS OF CULTURE MEDIUM TO TUBE NUMBER 2
AND 4.0 MILLILITERS OF MEDIUM TO TUBES 3, 4, AND 5.
USE A MICROPIPETTE TO REMOVE 0.5 MILLILITERS OF THE CELL SUSPENSION FROM TUBE NUMBER 1
AND TRANSFER IT TO TUBE NUMBER 2 FOR A 1:10 DILUTION.
THE CELL CONCENTRATION OF TUBE NUMBER 2 IS NOW 1,000 CELLS PER MILLILITER.
LET'S DILUTE THE CONCENTRATION AGAIN.
TRANSFER 1 MILLILITER FROM TUBE NUMBER 2 TO TUBE NUMBER 3 FOR A 1:5 DILUTION AND A CONCENTRATION
OF 200 CELLS-PER-MILLILITER.
TRANSFER 1 MILLILITER FROM TUBE NUMBER 3 TO TUBE NUMBER 4 FOR A 1:5 DILUTION AND A CONCENTRATION
OF 40 CELLS-PER-MILLILITER.
FINALLY TRANSFER 1 MILLILITER FROM TUBE NUMBER 4 TO TUBE NUMBER 5 FOR A 1:5 DILUTION YIELDING
8 CELLS-PER-MILLILITER.
LET'S GO BACK TO THE LAB TO COMPLETE THE SERIAL DILUTION.
ADD THE APPROPRIATE AMOUNT OF CULTURE MEDIUM TO TUBE NUMBER 1.
BASED ON THE CONCENTRATION OF OUR ORIGINAL CELL SUSPENSION, WE'VE CALCULATED THE VOLUME
OF CELLS AND CULTURE MEDIUM WE NEED IN ORDER TO HAVE A FINAL CONCENTRATION OF 10,000 CELLS
PER MILLILITER IN A FINAL VOLUME OF 5 MILLILITERS.
CLICK THE LINK ON YOUR SCREEN TO LEARN MORE ABOUT THESE CALCULATIONS.
NOW ADD 4.5 MILLILITERS OF MEDIUM TO TUBE NUMBER 2.
AND ADD 4 MILLILITERS OF MEDIUM TO TUBES 3, 4, AND 5.
USING THE PREVIOUS CALCULATION AGAIN, TRANSFER ENOUGH CELLS TO TUBE NUMBER 1 SO THAT THE
FINAL CONCENTRATION IS 10,000 CELLS-PER-MILLILITER.
AS YOU PERFORM EACH DILUTION, BE SURE TO MIX THE CELLS REPEATEDLY BY PIPETTING UP AND DOWN
SEVERAL TIMES.
THIS WILL HELP TO KEEP THE DILUTED CELLS UNIFORMLY SUSPENDED IN THE MEDIUM.
TUBE NUMBER 1 CONTAINS 10,000 CELLS-PER-MILLILITER.
REMOVE 0.5 MILLILITERS FROM TUBE NUMBER 1 AND TRANSFER IT TO TUBE NUMBER 2 FOR A 1:10
DILUTION.
THE CONCENTRATION OF TUBE NUMBER 2 IS NOW 1,000 CELLS-PER-MILLILITER.
TRANSFER 1 MILLILITER FROM TUBE NUMBER 2 TO TUBE NUMBER 3 FOR A 1:5 DILUTION.
THE CONCENTRATION OF TUBE NUMBER 3 IS NOW 200 CELLS-PER-MILLILITER.
TRANSFER 1 MILLILITER FROM TUBE NUMBER 3 TO TUBE NUMBER 4 FOR A 1:5 DILUTION.
THE CONCENTRATION OF TUBE NUMBER 4 IS NOW 40 CELLS-PER-MILLILITER.
LASTLY, TRANSFER 1 MILLILITER FROM TUBE NUMBER 4 TO TUBE NUMBER 5 FOR A 1:5 DILUTION WITH
A CONCENTRATION 8 CELLS-PER-MILLILITER.
NOW WE'RE READY TO SEED THE 6-WELL PLATE.
TRANSFER 2 MILLILITERS FROM EACH TUBE TO ITS CORRESPONDING WELL.
SINCE WE HAVE 5 TUBES, WE'LL ONLY BE USING 5 OF THE 6 WELLS.
PLACE THE 6-WELL PLATE IN THE INCUBATOR AND ALLOW 1 TO 3 WEEKS FOR COLONIES TO FORM.