Flow cytometry-troubleshoot - (Jun/06/2008 )

I've been struggling to use FACS and need some advice. I'm having trouble gating teh cells and also when I gate,I get a very weak signal. I'm using a protocol established successfully by an ex-colleague. But I cannot replicate teh experiment. I'm using the following antibodies for my staining. Please advice of any error.

1) cell line: human Hepatocellular carcinoma (HCC) cell line
2) Primary antibody: mouse anti-human antibody
3) secondary antibody: goat anti-mouse IgG (FITC)
4) control: mouse IgG with hCC cell. Now, if my cell line is of human origin, I should be using human IgG right?

Thanks
SF

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Hi SF,

Waht is your surface marker for your cell line taht you looking at?
how do you prepare your cell staining?
Do you stimulate your cell before staining?

hadrian

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Hi,

Well trypsinization of cells on the same day as staining results in cells clumping. So, I trypsinize my cells 24 hours before stainng and on teh day of staining them, I use only 5mM EDTA to dissociate the cells.

1. I stain the cells (2million cells suspended in 100ul PBS) with my antibody (0.5ug) for 30min in 4deg,
2. wash with 1ml PBS and spin at 300g for 10mins at 4deg.
3. remvove supernatant and add 100ul PBS and secondary antibody
4. Inubate for 30min in dark at 4deg
5. wash with 1ml PBS and spin at 300g for 10mins.
6. Remove supernatant in resuspend cells in 1ml PBS
7. ready for staining

I perform steps 1-6 in 1.5ml eppendorf tbes and transfer the 1ml suspension in step 6 to a flow cytometry tubes. Do you think the eppendorf tubes may be having some effect Shall I perform the staining form step 1 in the flow cytometry glass tubes?

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What concentraton of primary and secondary antibody would you recommen for FACS. I have tried 0.5ug of primary antibody and 15ug secondary antibody ( think this is too much?). I'm using a protocol from an ex-colleague who left. But the concetration of the secondary antibody does not seem right, does it?

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Sorry let me correct my above statement.
What concentraton of primary and secondary antibody would you recommen for FACS. I have tried 0.5ug of primary antibody and for my secondary antibody I use 1:10 dilution of teh total volume which is 10ul of secondary antibody (1.5mg/ml) added to 100ul cell suspension. I'm using a protocol from an ex-colleague who left.

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Swarat,

I do not do FCM with human cell line. I am not sure of your question if eppendorf will affect your results. I have always used FACS tubes.

You have been using PBS for washing as well as suspending cells. I use FACS Buffer that has Sodium Azide and BSA also.

About the concentration, it depends on the secondary antibody U r using. Please refer to the data sheet given with the packet for number of cells in suspension and amount of antibody. And, it is advisable to titrate yourself to meet your needs. Isn't it possible to consult with your ex-colleague about the protocol. If he has been doing it well then he should be able to give you some hint to start with.

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I've been told to use complete culture medium (or Hanks balanced sal solution containing NaN3, BSA). followng is the FACS staining protocol suggested to me by my PI. My concern is step 1 and 2. Should I resuspend cells (2x10 to the power 6) in 100ul of 4°C culture medium and take 50ul of that for staining instead of resuspening in 1ml and taking just 50ul from that? My concern is that taking 50ul cell suspension 1ml cell suspension won't give me enough cells, even though I'm just analysing 10,000 events on the FACS. I my concern justified? In the past I was staining 2x10 to the power 6 cells and was told it was too many.

Antibody staining for FACS

1. Resuspend cells (2x10 to the power 6) in 1ml of 4°C culture medium.
2. Add 50ul cell suspension (10 to the power 6 cells) to a tube containing ug primary antibody and mix gently with a 1ml pipette tip. Incubate at 4°C in dark for 30mins.
3. Wash cells by adding 2ml of 4°C culture medium. Centrifuge cell suspension 6min at 300Xg, at 4°C.
4. Discard supernatant
5. Resuspend cell pellet in 50ul culture medium and add ug secondary antibody. Mix gently with a 1ml pipette tip. Incubate at 4°C in dark for 30mins.
6. Wash cells by adding 2ml of 4°C culture medium. Centrifuge cell suspension 6 min at 300Xg, 4°C.
7. Resuspend stained cell pellets in 400ul of 4°C culture medium, Keep cell suspension on ice until analyzed by flow cytmetry.

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Regarding steps 1 and 2.

2 million cells is too much. Your PI suggested 0.1 million cells per tube which should be good. That is lot of cells. You should try, titrate and see.

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Thanks for your suggestion.

So, I will be resuspending 2 million cells in 1ml culture medium. And i will be only staining 50ul of the cell suspension. Now I have to dilute my IgG antibody (control). Do I dilute in PBS or culture medum?

Thanks
SF

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Whichever U r using to suspend cells, I guess. I have always used FACS Buffer but if u r using culture medium for suspending cells, it must be for some reason. Why is it?

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