Hello everyone, I am starting to culture THP-1 cells but I need some guidance.
I am sorry or all the newbie questions, but the former student who started the culture in our lab, left without teaching anyone and doesn't want to help me.
So, here are my main doubts:
1) Regarding PMA differentiation: I've read about 15 papers and each and every one of them uses different concentrations of PMA without much consensus. One thing that I found was that PMA can induce non-specific gene expression, so I will use low PMA concentrations. Maybe 5 or 10 ng.
2)My cells don't seem to grow as fast as they should. I've spoke to a colleague from another University and her THP-1 grow quite fast and change the pH of the medium, changing its color. Mine haven't changed the pH in 2 weeks!
3)I don't know for sure how to count cells in the counting chamber. Could anyone please send me pictures about their counting process. I know that this is a really dumb question, but i have different sized cells and I don't know which i should count.
4)About the amount of cells to plate: I am planning to infect the monocyte derived macrophages with M. tuberculosis, to study their RNA expression inside the cell among other goals. In general, the MOI of 1:1 or 1:5 are the most common ones to use. Considering the amount of cells, is 5x10^5 the only option? I don't know if I will be able to have enough cells to plate.
I am really sorry for asking such "beginner" questions, but that is really what I am now.
Thank you very much for your attention
Hi João,
I have been working with THP-1 cells for over 3 years now. Trust me they gave me a hard time too at the start. To anwer your questions:
1) Regarding PMA differentiation: I use 25 ng/ml for 72 hrs to differentiate them to M0 types and rest them for atleast 24h again before treating them.
2) My cells don't seem to grow as fast as they should. They do grow really fast, I spelt them twice a week to seed them around 3x10^5 /ml. Make sure you are using the ATCC modified RPMI-1640 (https://www.lgcstandards-atcc.org/products/all/30-2001.aspx) + heat inactivated FBS
3) We have a cell counter in our lab which is very handy but I have counted them manually as well and the cells are round and similar in shape. The cells look like this (https://www.lgcstandards-atcc.org/~/media/Attachments/F/4/5/D/2001.ashx ). If you see a different shaped cells, might be they are differentiating??!! Also, make sure to use non-adeherent flasks for seeding and mainating the cultures.
4) I have had culture grow to 1x10^6 / ml if i needed more cells, ATCC recommends not to let them grow over 8x10^5 cells /ml. Some times if i need to more cells, i grow them in two flasks.
Good luck
Feroz
We use THP-1 (4x106 cells) in a T25 flask placed horizontally and add PMA at 40 nM. They all adhere in 24h and become well-differentiated following 72h. THP-1 cells slowly proliferate when they are first thawed then they like to be a bit crowded for first days. Adding non-essential amino acids can help. It is hard to follow if they are alive just looking at their morphology, better you take a sample from the culture and count them after mixing them with trypan blue or other viability dye.
Hello! Thank you both for your answers! It sure will help me a lot.
Sorry for the delay answering. But here are my considerations:
Feroz Ahmad,
About the FBS inactivation: I haven't found consensus while searching for macrophage infection. Do you think I should inactivate my FBS then? My colleagues wich work with cytotoxicity don't inactivate their FBS, so I followed the example. I'm not sure about this.
Regarding PMA differentiation: I will make a pilot type study to test different PMA concentrations and choose the one I found the best. In the article I cited before (https://www.ncbi.nlm.nih.gov/pubmed/17334670), the lowest concentration of PMA which caused good differentiation and cell attachment was 5ng. So I'll try to test the following concentrations: 5ng, 10ng and 25 ng for different exposure times.
Güneş Esendağlı
I've done Trypan blue treatment and they seem to be viable (>90%). I don't know yet why they are multiplying "slowly". I guess that from now on the cells will start to do better.
Also, I am attaching some pictures i've took today to example my doubts.
The first picture shows the cells from my culture flask in 100, 200 and 400 x increase in the microscope. Also, today (for the first time) my medium changed color, from red to orange/dark yellow.
The second picture are some of the cells I'm having trouble counting.
A - Green circle: cells I consider as "good cells"; Red circle: (?) cell debris, cell residues?
B - Green circle: cells I consider as "good cells" and count; Red circle: Small sized cells, which I don't know if I should count.
C - Green circle: cells I consider as "good cells" and count; Red circle: Different shaped "cells", which I don't know if I should count.
Thank you very much for your time and willingness to help!
João Vítor
Yes, I think it is necessary to inactivate the serum when working with immune cells to inactivate the complement system. You can buy the heat inactivated FBS now most of the suppliers sell it now or you can heat inactivate it by Heating serum to 56°C for 30 minutes.
Hello!What's around THP-1 cells in the pictures?Debris or microorganism?Those dark yellow one.
Hi! anyone here has had good success nucleofecting ThP1 and would like to share your protocol? We have been trying with the neon system without much success. We do not differentiate them because we need to expand after nucleofection.
Thanks!
We cultured THP-1, always have a lot of cell debris, how to address this problem?
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