Scribd
Upload
12 views2 pages

PBL Lab Hand Out

Uploaded by

bsf79kxmms
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
12 views2 pages

PBL Lab Hand Out

Uploaded by

bsf79kxmms
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
You are on page 1/ 2

Time-dependent uptake gold nanoparticles (GNPs) by cancer cells

Goal: To quantify and compare the results of gold nanoparticles (GNP) uptake by brain cancer
(glioblastoma) cells after incubation for different time points. Relevance to nanomedicine: Radiotherapy
and photothermal therapy are examples of cancer treatment. GNPs enhance the damaging
absorbance effects of high-energy photons in these treatments or in other words, make the cancer cells
more sensitive. It is therefore crucial to understand how many GNPs (i.e., dosage) to use to get the
best possible results. There are various factors that help determine the proper dosage of GNPs. Some
basic factors that determine cellular uptake include size of the nanoparticle, time of incubation, material
of the
nanoparticle etc. In this lab we will test the effect of time of incubation of 24 nm sized GNPs on
cellular uptake.

Lecture context: We discussed various mechanisms through which cells take in nanoparticles in
lecture 3. Please refer to the lecture again before coming to the lab. Next, one of the virtual
assignments you did was cell culture simulation. Please refer to that assignment as well before coming
to the lab.

Additional reference: Please watch the following YouTube video about biosafety cabinets.
https://www.youtube.com/watch?v=HDKqOMybf4A

Method
Reagents provided:
• Solution A: 15 ml of cell culture media
• Solution B: 2ml solution containing GNPs of 24 nm.

Step 1: Transfer 3 ml of cell culture media (Solution A) to a cuvette. This must be performed in the
biosafety cabinet. Use this to blank the spectrophotometer (i.e., this is the absorbance of your solution
without GNPs).

Step 2: In the biosafety cabinet, add 2 ml of Solution B to 10 ml of solution A in a 14ml tube (let’s
call this Solution C). Put the lid on the tube and tighten it. Mix the solution contents by inverting the tube
3 times. Transfer 3 ml of this into a cuvette and measure the absorbance between 300 to 700nm, using
the spectrophotometer. You will see an absorbance peak between 500 to 600nm as shown in the figure
on the right. This value is indicative of the amount of GNPs you are starting out with. Note this
absorbance
as GNPs in media at time point “0”.

Step 3: Observe the flask with cancer cells under the microscope to make sure your flask contains
cancer cells. In the biosafety cabinet, add 10ml of the Solution C to your flask that has cancer cells.
Move the flask to the incubator where the cells will be provided with optimal conditions to function.
Leave this flask in the incubator for the time assigned to your group.

Step 5: At the end of the incubation time, please take your flask to the biosafety cabinet and transfer
the media from the flask to a 14ml tube. Put the lid on the tube and tighten it. Mix the solution contents
by inverting the tube 3 times. Transfer 3 ml of this into a cuvette and measure the absorbance between
300 to 700nm, using the spectrophotometer. You will see an absorbance peak between 500 to 600nm.
This is absorption at time point “t”.

Step 6: Calculate % uptake of GNPs by cancer cells for your assigned time of incubation as shown
below: % GNP uptake by cancer cells = (Absorbance at time point 0) - (Absorbance at time point t) x
100
(Absorbance at time point 0)
** For times of incubation that fall outside the class time, a teaching assistant will collect the samples
and provide the readings to you.

Lab report (1 page max)

• Submit 1 lab report per group and include the names of students who participated in generating this
report.

• If you think someone in your group did not contribute significantly to the generation of this report,
please do not include the names of those students (just because they were present in the lab with you).

• Hard copies of this report are due by Sept 25th, in class. Late submissions will not be accepted.

Format: Any standard type of font at point 12, double spaced.

Content:
1. In 1 or 2 paragraphs answer the following questions:

• What is the purpose of this experiment.


• What happened to cellular uptake with increasing time of incubation?
• Based on the types of cell entry mechanisms discussed in lecture 5, what type of cell entry
mechanism
do you think would have been used to take up the GNPs and why?
• Can you make the conclusion you made in response to question 2 above? Why or why not?

2. Draw a bar graph showing relative absorption at time points “0” and “t”
3. Draw a bar graph just like (2) above but also include absorbance at time points t from all groups
(your
instructor will upload data from all groups on canvas).

All graphs should have the X and Y axis labeled appropriately.

You might also like