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Lab 3: Cell Growth Curve
This lab allowed me to test the viability of my cell culture through the use of Trypan Blue. Furthermore, this lab allowed me to calculated the population doubling of the HEK 293 cells.
Materials
70% Confluency
HEK 293 Cells
TE and MEM media aliquots
12x 8cm^2 TC Plates (35mm)
Pipetman Pipettes
0.5-10ul
200-1000ul
MEM Media
Trypsin EDTA
15ml Tubes
1x 10cm Tissue Culture Plate
Hemocytometer
0.4% Trypan Blue
1.5ml microfuge tubes
Prelab Preparation
Please refer to lab 1: Making MEM media for Biosafety hood and water bath setup
Cell Preparation
1:8 Split
1:8 Split
The protocol that was used in Lab 2 to split cells was also used in this lab. A 1:8 split was accomplished by adding 1ml of the 8ml resuspended cell solution into two fresh culture plates with 10ml of MEM media. The cells pictured were at a starting confluency of about 85%.
Viability Test: Trypan Blue
Trypan Blue is utilized to test the viability of cells because it allows for the differentiation between living cells and cells that are apoptotic. This occurs due to the fact that Trypan blue is toxic and living cells pump the dye out of the cytoplasm while dead cells retain the dye and show up blue under a microscope.
With the remainder of the 7ml cell suspension the viability of the cells was tested. The cells were vortexed by flicking and 50ul of the cell suspension was drawn and placed into a 1.5ml microtube. After 200ul of Trypan Blue was added to the same microtube and were mixed together.
Viability/Cell Count
With the cells suspended in the Trypan Blue, 10ul were added to each wedge of the Hemocytometer and the cells were counted. Both stained and unstained. Stained cells shown in picture below. Live, unstained cells show up clear and look like air bubbles.
The picture on the right shows cells mixed with Trypan Blue and viable cells can be seen. Image was take with a phase contrast microscope.
Day0 cal.pdf100K Cells Per Plate Calculation (Day 0)
100K Cells Per Plate Calculation (Day 0)
The Day 0 cell count was conducted at 12:30pm and a total of 34 cells were counted on the hemocytometer. The number of cells was then multiplied by 0.7 to account for dying cells. The number of cells in both the 1.5ml tube and the 15ml tube were also derived in order to calculate if enough cells are present for 12 culture plates at 100k cells per each. 20.8 plates were possible and each plate received 336ul of cell suspension.
12x 35mm plates were labeled with the corresponding day. Two for each day were labeled Day 1-Day 6. Then 1ml of MEM media was added to each plate, as pictured above.
336ul of cell suspension were then added to each of the twelve 35mm plates to achieve 100,000 cells per plate. This was based on the calculations shown earlier.
With the addition of the MEM media and 100,000 cells to each plate, they were then transferred to the 37C incubator. Culture plates were placed in appropriate order based on the days for easy access.
Daily Cell Count Procedure
For each day following the set up of the twelve plates, the cell counts were conducted outside of the hood. Before counting cells, the MEM media was removed using the manual pasture pipette. 0.5ml of the aliquoted Trypsin EDTA was added to the plates and then they were transferred back to the incubator for 5 minutes. After incubation, 0.5ml of MEM media was added to the plate to neutralize the trypsin. Using a pipetman, 10ul of the cell mixture from the plate was then added to each wedge of a clean hemocytometer.
Day 1
Day 1
5% confluency
27.83 hours elapsed
80cells/ 8 quadrants= 10 cell average
10x10^4= 100,000cells/ml
100,000cells/ml /8= 12,500cells/cm2
Day 2
Day 2
15% confluency
48.18 hours elapsed
82cells/ 8 quadrants= 10.25 cell average
10x10^4= 102,500cells/ml
102,500cells/ml /8= 12,812cells/cm2
Day 3
Day 3
40% confluency
72.88 hours elapsed
327cells/ 8 quadrants= 40.875 cell average
40.875x10^4= 408,750cells/ml
408,750cells/ml /8=
51,093.75cells/cm2
Day 4
Day 4
60% confluency
96.72 hours elapsed
330cells/ 8 quadrants= 41.25 cell average
41.25x10^4= 462,500cells/ml
462,500cells/ml /8=
51,562.5cells/cm2
Day 5
Day 5
80% confluency
121.33 hours elapsed
443cells/ 8 quadrants= 55.375 cell average
55.375x10^4= 553,750cells/ml
553,750cells/ml /8=
69,218cells/cm2
Day 6
Day 6
95% confluency
145.83 hours elapsed
1192cells/ 8 quadrants=149 cell average
149x10^4= 1,490,000cells/ml
1,490,000cells/ml /8=
186,250cells/cm2
Cell Growth Curve Graph
This figure is the representation of the cell growth curve of HEK 293 cells. The X-axis is the number of hours that have elapsed and the Y-axis represents the number of cells on a logarithmic scale.
Plot of Population Doubling Time (PDT)
T= 24.5hrs
Xb= 69,218 cells
Xe= 186,250 cells
PDT= 17.15hours
PDT = (24.5) ln (2) / ln (186,250 / 69,218)= 17.15
Population Doubling Time = 17 hours
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