Obtain fresh mouse lung tissue and rinse with PBS to remove blood or debris by perfusing 3 ml PBS through the trachea or main bronchi.
Place the lung tissue on a cell strainer 70 microns on a 50 ml falcon tube.
Gently mash them with a plunger to extract the cells and tissue fragments.
Rinse the cell strainer with sterile PBS to collect any remaining tissue pieces. A Pro tip: Lift the cell strainer a bit from time to time to improve PBS drainage.
Centrifuge the collected cells at 2000 RPM for 7 minutes to remove red blood cells.
Implement red blood cell lysis if needed (Sigma Product No. R 7757):
Add 1 ml of buffer to the cell pellet.
Gently mix for 1 minute.
Dilute the buffer with 15 ml of PBS.
Centrifuge at 2000 RPM for 7 minutes and decant the supernatant.
Resuspend the cell pellet in full media (DMEM, 10% FCS, 0.5 % Pen-Strep, 1% glutamine, 1% NEAA, 1% pyruvate,1% fungizone) at a density of 200,000 cells per 75 microliters with 37.5 microliters of supernatant from HL-60 cells that are enriched with growth factors.
Plate the lung cells onto a 96-well plate and incubate for 72 hours to give time for cells to attach and secrete the ECM.
Preparation of Lung ECM:
After 72 hours of incubation, remove the media from each well.
Wash each well with 100 microliters of sterile PBS to remove any cellular debris.
Place 100 microliters of sterile distilled water (DDW) into each well to decellularize the wells for 30 minutes.
Remove the DDW and add 37.5 microliters of fresh ______ full media to each well. (depending on the cell line)
Seeding of Cells:
Prepare a cell suspension of 20,000 cells in 37.5 microliters of ____ full media. (depending on the cell line)
Plate the cell suspension in quadruplicates on a 96-well plate containing either lung-decellularized ECM or plastic.
Switch ________ full media to serum-free media with or without treatments after overnight incubation. (depending on the cell line)
Incubate the plate at 37°C and 5% CO2 for ____ hours. Recommendation (48-72 hours)
Analysis:
After the 96-hour incubation period, assess the cell viability and proliferation using standard assays such as CCK-8, and collect supernatants.
Compare the results of the cells cultured on lung decellularized ECM with those cultured on plastic to determine any differences in cell behavior.
For 96-well plates, you can download a protocol template:
Cultured tumor cells (200,000 cells in 40 µL saline per mouse)
1.5 mL Eppendorf tubes
Sterile 27-32 G needles (A Pro tip: Adjust needle size by checking if counted cells stay alive in vitro after drawing them and dispensing in plate vs. the exact number routinely seeded – if not, lower the G of your needle)
1 mL syringe
Isoflurane
Q-tips (used for iodine solution and eye cream applications; to give an extra stretch to the skin)
Iodine solution
Balb/c mice
An eye cream that prevents dryness
Gauze Pads
Preparation of Cells:
Mix the tumor cell suspension well but gently.
Draw the cells without the needle attached.
Attach the needle (to remove bubbles) and adjust the tip to align with the scale. A Pro tip: If you want to eliminate bubbles, draw more L than you need. You’ll need to draw some air into the syringe to increase the distance between the fluid level and the tip. To remove bubbles, flick the syringe. Empty all air and dead space until you see the first drop at the tip of the needle. Make sure you leave only the volume necessary for injection.
Preparation of Mice:
Weigh and number the mice.
Anesthetize the mice with isoflurane.
Cover the eyes of the mice with protective ointment to prevent dry eyes.
Lay the mouse on the gauze pad before shaving. A Pro tip: It is easier to collect hair with gauze pad.
Shave the mice.
Remove all the hair with the gauze pad.
With a Q-tip dipped in an iodine solution, scrub the injection area once. A Pro tip: Iodine solution stains all the skin except the nipple area, which makes it easier to identify injection sites.
Injection Technique:
Identify the fourth abdominal mammary gland.
Lift the skin (3-4 mm) near the fourth mammary gland nipple area while placing the forceps (1-2 mm above the nipple) before injecting. A Pro tip: Some mice may have stretchy skin that obstructs vision. To improve access, ask your colleague to gently stretch the skin above and below the injection site using Q-tips.
Insert the needle (2-3 mm) from the side at an angle of 180° under the nipple area. The tip should be inserted with an additional 0.5-1 mm. A Pro tip: Once the needle has passed the skin barrier and been positioned at the correct depth, lift the tip of the needle with the nipple. The cells are injected beneath the nipple.
Inject 40 µL (not more than 50 µL) of the tumor cell suspension.
Remove the needle slowly to avoid leakage.
Injecting properly will form a visible, palpable round bump that disappears without leaking any cells.
Observe the animal after waking up from anesthesia in its follow-up cage.
Return the animal to its original cage.
Tumor growth and development should be monitored twice a week in the mice. Depending on the cell type and number injected, tumors are palpable approximately seven days after injection and can be measured with calipers. It should take 2-3 weeks for tumors to form a tumor that can be removed easily.
It is highly recommended that you watch this video, even though the injection technique is different, in order to see how the fourth mammary gland nipple area is identified and what the bump looks like.
BSA (bovine serum albumin) standard solution stored with a concentration of 1.4 mg/ml
Bradford reagent (dye-binding reagent)
Test samples containing protein
96 well Microplate
Spectrophotometer
Procedure:
Prepare the BSA standard solutions of different concentrations (e.g., 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 µg/µL) by diluting the BSA stock solution with distilled water as follows.
BSA standard preparation for working concentration 1mg/ml
45 µL (BSA 1.4 mg/ml in DDW) +18 µL DDW
Final BSA Concentration µg/µL
Volume BSA from 1 mg/ml (µL)
Volume DDW (µL)
0
0
25
0.05
1.25
23.75
0.1
2.5
22.5
0.2
5
20
0.3
7.5
17.5
0.4
10
15
0.5
12.5
12.5
0.6
15
10
Add 10 µL of each BSA standard solution to the 96-well microplate in duplicates.
Prepare the Lysis Buffer by mixing 270 µL with 30 µL of Protease inhibitors (Sigma Cat. No S8820, 10x) for one lung tissue measuring approximately 2×2 mm. The ratio should be 1:10. Calculate the required volumes based on the number of specimens:
N of specimens x 300 µL = Final Volume
N of specimens x 30 µL = Required volume of protease inhibitors
Final Volume PBS = Final volume – Required volume of protease inhibitors
Sonicate specimens on ice using short pulses not exceeding 5 seconds.
Incubate specimens on ice for 30 minutes to cool down.
Centrifuge at 12,000 RPM for 15 minutes.
Collect fresh supernatant in a separate Eppendorf tube.
Data processing can be time-consuming, but it’s an essential skill. If you don’t handle repetitive tasks smartly, they can drag on. Plus, knowing how to manage these tasks easier will save us time for more meaningful things or give us a minute to relax.
For example, during my thesis, I learned how to make some tasks on the computer faster and more automated. As a result, I could focus on more relevant jobs, invest more time in hobbies I enjoyed, and even write this blog.
The lab’s most valuable resource is time, and using it wisely is the key to success. So, in this article, I wanted to share eight tips that I’ve found helpful to make repetitive tasks easier:
“He who can copy can do” -Leonardo da Vinci.
When it comes to working fast, using prepared templates and protocols is essential. Let’s say we are repeating an experiment a third time while using the same conditions; the number of samples is different. We can write the protocol from the beginning, copy it from our previous experiment, and make small changes.
The best way to accomplish this is using an electronic lab notebook, such as Notability. With this app, you can upload separate protocols in the same notebook, add hand-written comments, mark progress with an Apple pen, and, most importantly, use the lasso tool. This tool allows me to copy and paste previous hand-written calculations and to update relevant information, such as the number of wells and the outcome of my calculations.
For a quick tutorial on using the lasso tool cleverly, I’d recommend watching this short video – it’s straightforward.
Alternatively, you can prepare a template in excel for your calculations. Or, you can check out the collection of electronic protocols for typical assays used in research (work in progress)here.
Combine fun and work
We are tempted to passively perform repetitive tasks and to divide our focus. While outlining irregular wound scratch assays, I sometimes watch TV or do something else. When I outline them, the program I use mustn’t freeze or lag, the process itself should be as smooth as possible, and mistakes should be easy to fix. It works for tasks that do not require full attention and are easy to fix if something goes wrong. To outline irregular migration assays, which lack contrast to the background, investing in an Apple pen was extremely valuable.
The first thing to optimize your focus is to identify tasks requiring a high level of attention. These tasks demand thinking or cost a lot of resources (time, materials) to fix if a mistake has been made. As a result, I have developed several ‘hacks’ that have helped me stay focused on what I’m supposed to be doing. Firstly, you can track your progress on known protocols even when you are tired or multitask between experiments by marking ticks as you proceed. Changing the alignment of test tubes after working with them is another trick. A task that is not recorded on paper is not completed. Successful focus begins with a break during which you wind up and satisfy your basic needs.
Whatever you can batch process, do it
In the world of computers, there’s this thing called ‘macro,’ which enables you to batch process almost anything. I have a video about this, and if you want to find out more, I have a pretty awesome guidebook about batch processing in image j, excel (in progress).
The idea behind macro is that we don’t push buttons or icons repetitively to perform the exact same task on the program. Instead, we teach a computer what steps to do, which files to process, and where and how to export outcomes.
And if you’ve had that experience where you’ve pressed a thousand times the same three buttons on the program, and you’ve entirely started to feel like a robot, that’s just because you haven’t given this task yet to macro, an actual robot that should have been doing this in the first place.
Save time by investing in time-saving skills.
There is something about the word programming that can instantly induce anxiety for some people. It’s especially true when we’re just starting something, and we don’t know what we’ll be able to achieve. At the start of any learning, we need an injection of positivity, simplicity, and instant results to see whether it is worth our efforts. But if we can see that these results are applicable and save us time, like understanding basic programming to batch process our data, we should dive into it.
Whether you want to batch process your data using image j, Excel, or R or simply survive your biostatistics class, here is a basic introduction to programming.
There is no need to do everything on your own
As my friend advised, I should place my pride/fear of asking for help deep in the drawer and lock it. It can be retrieved after meeting people. There is a huge chance that someone has already faced your issue or has enough experience to handle it smarter, not harder. Indeed, dealing with people has its peculiarities, such as working on a collective project, but as the African proverb goes, “Go alone if you want to go fast. If you want to go far, go together.” [1]. Do I always listen to my friends’ advice? My heart tells me it’s the right way to implement it, even if I’m not always convinced.
Choose bare-minimum time for implementing tasks.
Most of the time, I do tedious, repetitive tasks with a timer because starting is the biggest problem, and I keep putting it off. To make your lazy days’ tasks more manageable, pick the minimum time to complete them. Compounding effects work in the end, so sometimes it’s better to do a bit than nothing. If it worked in economics, it would work for you too.
Conclusions
Time is the only currency people steel; they quickly trade but never get it back. Your time in the lab or doing something you enjoy is worth more than being a robot pressing the buttons, so do repetitive tasks smarter, not harder.
[1] Whitby Andrew. Who first said: if you want to go fast, go alone; if you want to go far. Accessed on the Internet [3 Jan 2023]. Available via link
The core of any batch-processing code consists of the following:
Input directory
Output directory – I believe in most cases it is better to save the output in a separate directory, especially when working with more complex tasks. The example below, however, does not include an output directory.
Loop {} – indicates repetitive actions
File opening
Manipulations
File saving
This macro will process all the images in the selected source directory. It opens each image, converts it to 8-bit, applies “Smooth” and “Find Edges” filters, saves the processed image as TIFF with the prefix “processed_”, and closes the image. You can modify the steps according to your needs.
// Batch Processing Macro for ImageJ
dir = getDirectory("Choose Source Directory ");
list = getFileList(dir);
for (i=0; i<list.length; i++) {
opening
path = dir + list[i];
open(path);
//Manipulation
run("8-bit");
run("Smooth");
run("Find Edges");
// saving
saveAs("Tiff", dir + "processed_" + list[i]);
close();
}
To run a macro in ImageJ, follow these steps:
Open ImageJ.
Click the “Plugins” menu, then select “New” and then “Macro”. This will open the macro editor.
Copy and paste the macro code into the editor window.
Save the macro using the “File” menu and give it a descriptive name, such as “BatchProcessing.ijm”.
Close the macro editor.
Navigate to the “Plugins” menu, then select “Macros” and then “Run”. This will open the window where you need to find the directory and select in it the name of the macro you just saved. Choose Open.
The macro will run and process the images according to the steps defined in the code.
Note: You may need to modify the macro code to fit your specific needs and input the image
Where can this be applied?
You can automate any Image J task that requires repetitive button pressing. I’ve used it to merge two different fluorescence channels for all images. Additionally, I used it to add scale bars to pictures as well as extract coordinates for wound healing analysis.
If I have no experience in coding, what should I do?
Four options are available in this case:
To begin with, I strongly believe you should learn how to do it. The more you understand the basics of any coding language, the more you’ll understand how computers work. Furthermore, if you explain your needs clearly to the computer, it will solve your problem more effectively. Using documentation as your vocabulary source and adapting codes posted in forums will enable you to solve your problems.
To create your final puzzle, you can use the macro recorder in Image J to record your steps.
The codes can be borrowed from other forum members, such as the one here for Image J.
In addition, ChatGPT, which everyone is talking about nowadays, can help you generate code templates faster without no knowledge of coding. Based on my request, ChatGPT generated the code above. Check it out. It is completely free. You can get started right away by registering here.
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