Hello, do you mean your macrophages remain attached to the plate, can you see them under the microscope? or do they die upon detachment?

Usually you can detach THP-1 with trypsin-EDTA (or PBS-EDTA incubated 10-15 min on ice) + a very light sweep with a cell scraper, but it can affect cell viability. Maybe you could try TrypLE reagents from Life Technologies, it's very efficient for strongly adherent cells.

I remember seeing people using teflon plates to differenciate non-adherent macrophages, but I'm not convinced it's a good option.

Binbin Lee, you could use a special digestion solution Accutase (Reference PMID: 25226503).

You can also try adding Lidocaine. For cells that are difficult to digest, you can use 2% lidocaine to digest for 5-8 minutes and then discard it. You can also add culture medium and blow it, which has little effect on the cells.

Gentle detachment of adherent macrophages is achieved by enzymatic hydrolysis (Accutase I), combined with centrifugal collection, which is suitable for subsequent experimental operations such as transfection.

The specific method based on Accutase I for isolating THP-1 differentiated macrophages is as follows:

1. Preliminary preparation

- Preheating of reagents: Preheat Accutase I (cell digestion enzyme) and all culture media (such as RPMI-1640 (HY-K3004, MCE)) in a 37°C water bath.

- Confirmation of cell status: Ensure that THP-1 has differentiated into macrophages (adherent growth) in a culture medium containing 10 ng/ml PMA, and that the appropriate density is reached after 48 hours of culture (1.0-1.5×10⁷ cells in a 75 cm² culture flask, 2.5×10⁷ cells in a 150 cm² culture flask).

2. Cell detachment (key step)

- Removal of old culture medium: Aspirate the culture medium in the culture flask to avoid residual influence on digestion.

- Add Accutase I: Add the corresponding volume of Accutase I according to the size of the culture flask (6 mL for 75 cm² flask and 12 mL for 150 cm² flask) to ensure that the cell surface is completely covered.

- Incubate and digest: Incubate at 37°C for 30 minutes to allow the cells to fully detach. During this period, the cell morphology can be observed. If there are still attached cells, the culture flask can be gently blown or knocked to assist detachment (cell scrapers are prohibited to avoid damaging cell viability).

3. Cell collection and centrifugation

- Transfer cell suspension: Transfer the digested cell suspension to a 15 ml centrifuge tube.

- Centrifugal precipitation: Centrifuge at 300×g for 5 minutes at room temperature and discard the supernatant (Accutase I).

- Resuspend and count: Resuspend the cell pellet with 1 ml preheated RPMI medium and count the number of cells (make sure the concentration is suitable for subsequent experiments, such as transfection requires 2.0-2.5×10⁶ cells/sample).

Notes

- Digestion time control: Accutase I treatment time should not be too long to avoid cell damage; if using an automatic cell counter, you can count directly after digestion to simplify the steps.

- Cell viability protection: Avoid violent centrifugation or mechanical damage during the operation to ensure that the cell morphology is round (detached state) to maintain the activity of subsequent experiments (such as transfection efficiency).

The answer to this question comes from MedChemExpress (MCE) Technical Support.

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It's not clear if you are not recovering enough cells from culture (maybe caused by low differentiation of THP-1 caused by low levels or defective PMA), you are recovering cells but those are not viaible (your scraping is too agresive) or you are recovering cells but the rest are still in the plate (If the case is the last one, the anwser has already been provided here).