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Aiden Nguyen

Weekly Update (5/13/2016 - 5/19/2016)


1. Monitoring the Stability of Lip-G Samples
Table 1. Size of Lip-G Samples over 14 Days
Day
0
5
6
7
8
9
10
11
12
13
14

Sample 1 Effective Diameter,


nm
75.20
107.70
94.24
87.18
92.13
100.50
85.10
114.88
89.04
173.06
213.15

Sample 3 Effective Diameter,


nm
58.10
57.98
58.54
60.96
66.00
58.38
63.84
62.07
62.42
64.96
61.74

Stability of Lip-G Samples

Sample 1

Sample 3

Sample 3 is more stable than sample 1 because the formers size varies less.
The impurity in sample 1 (graphene particles) possibly causes the particles to
more easily aggregate.

2. Cell Culture
a. Medium Change
HTB Cells
1. In water bath, bring medium (RPMI 1640) to 37 oC

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2. Check cells under microscope
3. Pipette contents of flask into 15 mL tube
4. Centrifuge at 3500rpm for 5 min
5. Aspirate supernatant with Pasteur pipette
6. Add 1 ml of medium to cells and gently mix with pipette (resuspension)
7. Add content to flask
8. Add 10mL of medium using a glass pipette
9. Mix in figure 8 motion
10.Write down the date on flask
11.Check under microscope
12.Place the flask in the incubator and the medium in the fridge
RPE Cells
1.
2.
3.
4.
5.
6.
7.
8.

In water bath, bring medium (DMEM F12) to 37 oC


Check cells under microscope
Aspirate old medium with Pasteur pipette
Add 10mL of medium using a glass pipette
Mix in figure 8 motion
Write down the date on flask
Check under microscope
Place the flask in the incubator and the medium in the fridge

Observations
Quick cell proliferation in petri dishes
HTB cells are growing slowly
Noticeable cell proliferation in both flasks
Medium appeared orange indication of lower pH
b. Cell passaging
RPE Cells in Petri Dishes
In water bath, bring medium (RPMI 1640) to 37 oC
Check cells under microscope to see the confluency
Aspirate medium and add 5mL of DPBS to wash with gentle swirling
Aspirate DPBS
Add 2mL of trypsin and swirl gently; incubate for 5 min
Remove the 2 dishes from the incubator and tap on them to make sure cells
detach from the surface; check under microscope
7. Add 2mL of medium and transfer all the content to a 15mL centrifuge tube
8. Centrifuge cells at 3500rpm for 5 min, aspirate, and resuspend in 2 mL of
new medium
9. Add 10L of resuspended cells into 1.5l tube containing 90l of trypan blue
stain to make 10x dilution and mixed gently
10.Add 10L of dilution to hemocytometer (Cyto chip)
11.Count cells in 5 of 9 squares (four corners and center)
12.Add the resuspended cells into new labeled petri dishes (generation, date,
type of cells)
1.
2.
3.
4.
5.
6.

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13.Add 4.5mL of medium, and swirl gently
14.Check under microscope
15.Place in the incubator
Results
5/14 (only 1mL of medium was added)
Dish 1 3*105 cells/mL
Dish 2 5.4*105 cells/mL
Dish 3 4*105 cells/mL
Total: 1.24*106 cells in 6 petri dishes
5/16 (2mL of medium)
2 Dishes (1) 3.6*105 cells/mL (took out 2.1*105 cells for XTT)
2 Dishes (2) 6.4*105 cells/mL
2 Dishes (3) 3.8*105 cells/mL
Total: 2.2*106 cells in 11 petri dishes
5/19
3 petri dishes - 3mL of medium
2.6*105 cells/mL
Total: 7.8*105 cells in 6 petri dishes
Flask 2mL of medium
2.66*106 cells/mL
Total: 5.32*106 cells in 2 flasks
3. Assisted Edward in XTT Test for Lip-G Sample Using RPE Cells
Pre-Optimization Assay
1.
2.
3.
4.

Aspirate medium and add 5mL of DPBS to wash with gentle swirling
Aspirate DPBS
Add 2mL of trypsin and swirl gently; incubate for 5 min
Remove the 2 dishes from the incubator and tap on them to make sure cells
detach from the surface; check under microscope
5. Add 2mL of medium and transfer all the content to a 15mL centrifuge tube
6. Centrifuge cells at 3500rpm for 5 min, aspirate, and resuspend in 2 mL of
new medium
7. Add 10L of resuspended cells into 1.5l tube containing 90l of trypan blue
stain to make 10x dilution and mixed gently
8. Add 10L of dilution to hemocytometer (Cyto chip)
9. Count cells in 5 of 9 squares (four corners and center) to get a total of 18
cells.
18 x 10 (dilution) / 5 (# of squares) x 10,000 = 3.6*10 5 cells/mL
10.Volume of medium required in each cell
104/(3.6*105)*103 = 28 L
11.Add 28L in 21 wells (A1-C7), and top up to 100L with medium. Column 8
was medium alone.
12.Covered and placed in incubator at 2:42 pm on 5/16
13.Pipetted remaining unused cells into 3 petri dishes

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Sample Addition
Well
A

B
C

1
100L
Lip-G

2
80L
Lip-G

3
60L
Lip-G

4
40L
Lip-G

5
20L
Lip-G

6
10L
Lip-G

7
100L
sterile
water

Replicates
Top up to 100L with medium

Covered and placed in incubator at 3:15pm on 5/17


Finished Pre-Optimization Assay
1.
2.
3.
4.

Checked cells under microscope.


Added 50l of pre-mixed activated XTT reagent to each of 24 wells
Placed in incubator at 5:30 pm on 5/18
Read wavelengths at 475nm and 660nm on the Spectramax Plus 384 at 8:30
pm

4. Assisted Madison in DOPI Test

5.

Future Work
Continue to check the stability of Lip-G Samples
Monitor cell growth
Prepare more cells for XTT tests