Nov 27, 2025
L929 CONDITIONED MEDIUM (LCM)
- Joseph Chon1,2
- 1Bowdish Lab, McMaster University, Hamilton, ON, Canada;
- 2Updated: July 2018
- Parasitolab
Protocol Citation: Joseph Chon 2025. L929 CONDITIONED MEDIUM (LCM). protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7n1e3lwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 28, 2025
Last Modified: November 27, 2025
Protocol Integer ID: 225707
Keywords: bone marrow progenitor, expressing various myeloid surface marker, various myeloid surface marker, macrophage, l929 cell, macrophage colony, bone marrow, heterogeneous population of bone marrow, bmdm, cell, lcm, l929 conditioned medium, stimulating factor, bm8
Abstract
Macrophage colony stimulating factors (M-CSF) are secreted by L929 cells (ATCC) and promote bone marrow progenitors to differentiate into a heterogeneous population of bone marrow derived macrophages (BMDM) expressing various myeloid surface markers (CD11b, F4/80, BM8, CD31, CD68, CD11c, Ly6C, GR1)1
Image Attribution
Bowdish Lab logo (Bowdish Lab, McMaster University). Website: www.bowdish.ca
Guidelines
PROTOCOL
1. Thaw L929 fibroblasts stored in -140°C in a 37°C degree water bath for 2 minutes.
2. Pipette thawed L929 gently into a 50 mL falcon tube containing 20 mL of warm DMEM – 10.
3. Centrifuge the cell suspension at 1500 rpm for 5 minutes.
4. Resuspend the cell pellet in 5 mL DMEM – 10 media and pipette into a T25 tissue culture flask.
5. Incubate at 37°C, 5% CO2 overnight to grow to 70% Confluency.
6. Cells were lifted with 5 mL trypsin and pipetted into a 50 mL falcon tube containing 20 mL DMEM – 10 media. Cell suspension was centrifuged at 1500 rpm for 5 minutes.
7. Cell pellet was re-suspended in 15 mL DMEM-10 media and transferred to a T75 flask.
8. Incubate at 37°C, 5% CO2 overnight to grow to 70% Confluency.
9. Cells were lifted with 10 mL trypsin and pipetted into a 50 mL falcon tube containing 20 mL DMEM – 10 media. Cell suspension was centrifuged at 1500 rpm for 5 minutes.
10. Cell pellet was re-suspended in 25 mL DMEM-10 media and transferred to a T150 flask.
11. Cells were cultured for 2 days, reaching a confluency of 90% and split 1:5 into a new T175 flask. This process was repeated until 50 flasks were obtained.
12. Split L929 cells 1:5 into a new T175 flask containing 45 mL DMEM -10 and culture for 10 days at 37°C, 5% CO2.
13. The cell culture media was collected and centrifuged at 3000 rpm, 4°C. the media containing M-CSF was then filtered through a 0.45 µm filter and frozen at -27°C in 50 mL aliquots.
Materials
- L929 fibroblasts
- Water bath
- DMEM - 10
- Tissue culture flasks
- Incubator
- Trypsin [Invitrogen]
- Falcon tubes
- 0.45 µm filter
Troubleshooting
PROTOCOL
Thaw L929 fibroblasts stored in -140°C in a 37°C degree water bath for 2 minutes.
Pipette thawed L929 gently into a 50 mL falcon tube containing 20 mL of warm DMEM – 10.
Centrifuge the cell suspension at 1500 rpm for 5 minutes.
Resuspend the cell pellet in 5 mL DMEM – 10 media and pipette into a T25 tissue culture flask.
Incubate at 37°C, 5% CO2 overnight to grow to 70% Confluency.
Cells were lifted with 5 mL trypsin and pipetted into a 50 mL falcon tube containing 20 mL DMEM – 10 media. Cell suspension was centrifuged at 1500 rpm for 5 minutes.
Cell pellet was re-suspended in 15 mL DMEM-10 media and transferred to a T75 flask.
Incubate at 37°C, 5% CO2 overnight to grow to 70% Confluency.
Cells were lifted with 10 mL trypsin and pipetted into a 50 mL falcon tube containing 20 mL DMEM – 10 media. Cell suspension was centrifuged at 1500 rpm for 5 minutes.
Cell pellet was re-suspended in 25 mL DMEM-10 media and transferred to a T150 flask.
Cells were cultured for 2 days, reaching a confluency of 90% and split 1:5 into a new T175 flask. This process was repeated until 50 flasks were obtained.
Split L929 cells 1:5 into a new T175 flask containing 45 mL DMEM -10 and culture for 10 days at 37°C, 5% CO2.
The cell culture media was collected and centrifuged at 3000 rpm, 4°C. The media containing M-CSF was then filtered through a 0.45 µm filter and frozen at -27°C in 50 mL aliquots.
Protocol references
Bender AT, Ostenson CL, Giordano D, Beavo JA. Differentiation of human monocytes in vitro with granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor produces distinct changes in cGMP phosphodiesterase expression. Cell Signal. 2004;16(3):365-374.