Can anyone provide some advice on Zymosan phagocytosis assay (Cell BioLabs)?

27 January 2015 5 5K Report

I am working on developing a human macrophage differentiation protocol, and purchased the Zymosan phagocytosis assay kit from Cell BioLabs to test the functionality of my final differentiation product. The first attempt, however, went very poorly. Completely incoherent data from the plate reader, mostly cellular debris in the wells, etc. I think it is a matter of centrifuging the plates at a lower speed, but I am curious if anyone has any experienced tips about this technique. Please advise, and thank you!

Product Page: http://www.cellbiolabs.com/phagocytosis-assay-zymosan-substrate

Current Protocol:

~ Preparation: Poly-HEMA Coating (makes 20 wells in a 96-well plate)

Dilute 150mg Poly-HEMA in 1.5mL of 10 mM NaOH 95% EtOH (15 μL 1M NaOH, 60 μL dH2O, 1.425 mL 100% EtOH)

Incubate on orbital shaker O/N @ RT

Add 50 μL to each well of the 96-well plate and swirl thoroughly to ensure the surface is completely coated

Let air-dry O/N in the hood

Aspirate and wash with 100 μL PBS/well prior to use, can store at 4°C for up to 1 week prior to use wrapped in parafilm

~ Preparation: Transfer Macrophages to Poly-HEMA-Coated 96-well Plate

Harvest and re-suspend cells to 200K-1M cells/mL in culture medium

Plate 100 μL cell suspension into X wells of the 96-well plate

Incubate @ 37°C, 5% CO2 O/N

~ Preparation: Assay Reagents

Dilute blocking reagent 1:100 in 1X PBS/0.1% BSA, permeabilization solution 1:10 in 1X PBS, store @ 4°C

Immediately before use, dilute detection reagent 1:250 in 1X PBS

~ Phagocytosis Assay

Add 10 μL Zymosan suspension to each well, mixing well, incubate @ 37°C for 15’ to 2hrs, centrifuge at high speed, aspirate

Outside of the TC room, 200 μL ice cold 1X PBS wash

Fix with 100 μL fixation solution @ RT, 5’, centrifuge, aspirate

200 μL 1X PBS wash, 2X

Block with 100 μL 1X blocking solution @ RT, 60’ on orbital shaker, centrifuge, aspirate

200 μL 1X PBS wash, 3X

Permeabilize with 100 μL 1X perm. solution @ RT, 5’, centrifuge, aspirate - 200 μL 1X PBS wash, 2X

Detect Zymosan phagocytosis with 100 μL 1X detection reagent @ RT, 60’ on orbital shaker, centrifuge, aspirate

200 μL 1X PBS wash, 3X

Add 50 μL detection buffer @ RT, 10’ on orbital shaker

Add 100 μL substrate, incubate @ 37°C, 5’-20’

Add 50 μL stop solution, shake for 30’’

Read absorbance on plate reader @ 405 nm

Sagar Gaikwad Popular answer

Dear Chris,

The protocol seems to be accurate, the problem may be in the activation of macrophages.

Resting macrophages, shows very low phagocytic activity compared to activated macrophages. I would like to suggest that, activate the macrophages with immunogenic stimuli like LPS (100 ng/ml).

Compare Unstimulated and LPS stimulated macrophages for there phagocytic activity. In my experience LPS stimulation significantly increases phagocytosis.

Thanks

Sagar

Sagar Gaikwad

Dear Chris,

The protocol seems to be accurate, the problem may be in the activation of macrophages.

Resting macrophages, shows very low phagocytic activity compared to activated macrophages. I would like to suggest that, activate the macrophages with immunogenic stimuli like LPS (100 ng/ml).

Compare Unstimulated and LPS stimulated macrophages for there phagocytic activity. In my experience LPS stimulation significantly increases phagocytosis.

Thanks

Sagar

Chris Mcginnis

Good idea, thanks!

José Coelho Neto

Hello there Chris...

Are your Zymosan particles opsonized? It should improve engulfment rates...

You could also try increasing incubation temperature a couple of degrees after adding Zymosan, simmulating a fever... It may help improve phagocytosis as well...

I also agree with activating the macrophages first.

Hope it helps...

Coelho

Ana Camila Oliveira Souza

Hi there,

How many particles/macrophages are you using? This is a very important thing to consider, since you might be inducing cell death. That would explain why you have cell debris. A 30-100 particles/macrophage ratio should give you a better result.

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