Hello Anteneh,

Here is a protocol from James Bryers of University of Washington. Please google to find out relevant reference.

A. Preparation of stock solutions

NOTE: Solutions should be prepared fresh for each assay

1. 10 mL of 0.1 M sodium bicarbonate buffer, pH 9.0

i. Add 84 mg of sodium bicarbonate to 10 mL of DI water

ii. Adjust pH to 9.0 with sodium hydroxide

iii. Filter sterilize using syringe filter

2. 1 mL of 10 mg/ml FITC in anhydrous DMSO (prepare immediately before use in step B3)

i. Add 1 mL of anhydrous DMSO to 10 mg vial of FITC and vortex vigorously until dissolved

B. Preparation of FITC-labeled bacteria

1. Harvest bacterial cells by centrifugation at 10,000 x g and 4°C for 5 min.

2. Resuspend bacterial cells in 1 ml of 0.1 M sodium bicarbonate buffer. Measure the optical density at 600 nm from a 1:100 dilution of cell suspension in sodium bicarb. Dilute cells to 1010 cells/mL in a total of 1 mL sodium bicarb.

3. Add 0.2 μl of FITC stock solution to cell suspension and immediately vortex. Incubate cells in the dark with end-over-end rotation for 30 min at room temp.

4. Wash cells 4x with HBSS to remove unbound dye and resuspend in 1 mL of HBSS. Measure the optical density at 600 nm from a 1:100 dilution and resuspend cells to your desired concentration in either HBSS or medium (if using for phagocytosis assays).

Dear Anteneh,

The following is a general protocol:

GENERAL CELL STAINING PROTOCOL FOR FLOW CYTOMETRY

1) Except for cells grown in culture, cells obtained directly from tissues must first be resolved to

a single cell suspension by means of mechanical dissociation (mincing, grinding between the

ends of two frosted glass slides, dounce homogenization, steel mesh sieving, passage through

a fine-bore syringe tip etc.) or enzymatic digestion (collagenase, DNAse, neutral protease

disruption of stromal cells or pepsin digest of solid tissue). Mechanical dissociation of

spleen and thymus is effective in releasing lymphocytes but is often damaging towards

larger, blastic cells and usually leaves dendritic and stromal cells behind in the debris. These

cells are obtained through gentle enzymatic digestion.

Prior to staining, cells must then be purified by at least one of several procedures to enable

analysis on the flow cytometer without damage to the flow cell and permit staining without

excessive background. This will invariably include some combination of density gradient

centrifugation, positive or negative selection procedure (panning, complement-mediated cell

lysis) straining through nylon-mesh and/or red-cell lysis where necessary. (Prior to the BD

FACScan, earlier instruments mandated nylon mesh screening s the smaller flow cells on

these machines could easily become clogged by clumped or aggregated cells and often this

could take months and hundreds of dollars to repair!)

2) Wash purified cells 1X in staining buffer. (A suitable buffer will be isotonic and buffered to

neutrality, will cushion the cells against damage during centrifugation, block non-specific

staining, prevent capping of bound antibody and will block Fc receptor binding.)

e.g. formulations:

.05 M Tris buffered Saline pH 7.4 or: Phospate buffered Saline

1% normal sera of the animal for which 2% Bovine Serum Albumin

the primary antibody is derived or 0.1mg/ml 0.1% Azide

Ig of such animal. (will not block Fc receptor binding)

2% Fetal Calf Serum

0.1% Azide

3) Adjust concentration of cell suspension to at least 5 x 10

6 cells/ml. Cells should be greater

than 90% viable as determined by trypan blue exclusion. Determine # of samples necessary

for experiment and aliquot (1 x 10

5 – 1 x 10

6 cells/sample, preferably the higher value if

possible) to U bottom microtiter well or plastic centrifuge tube.

4a) For staining in microtiter plates. Spin plate for 3 minutes at 100 x g (usually 600 rpm) at

8o

C. Flick plate or remove fluid from wells by suction and immediately resuspend pellet in

100ul of 1st reagent, appropriately diluted to previously determined titration point in staining

buffer (primary antibody directly labeled (e.g. with FITC) if performing direct-labeling,

primary antibody biotinylated or unlabeled if performing indirect staining.) Incubate 30’ at

4o

C. (in the dark if fluorescent labeled)

5a) Wash 3X. (Spin, flick, resuspend in S.B.) After last wash, flick and resuspend in 100ul of

secondary reagent (e.g. FITC labeled Goat anti-mouse Ig or Avidin-FITC also pre-titered)

and repeat step 4 if performing indirect staining, otherwise simply resuspend in 100ul of

staining buffer and transfer to Falcon 2052 tube (this is the only tube that fits the FACScan)

containing 3-500ul of staining buffer. Keep samples on ice and perform FACS analysis. If

samples cannot be run immediately fix them in 1% formaldehyde or 3% paraformaldehyde in

PBS for 15 minutes at 4o

C, wash 1X in staining buffer (samples are left in fix by most

researchers but this writer has found a great deal of cell clumping without fixative removal),

store in fridge in the dark for later analysis.

4a) For staining in tubes. Centrifuge at 300 x g (usually 1000 rpm) for 5 minutes at 8o

C.

Carefully aspirate supernatant from cell pellet and resuspend pellet in 100ul of 1st reagent in

staining buffer as above. Incubate and wash as above for direct or indirect procedures.

Important Things to Remember:

1) Both primary and secondary antibodies or avidin reagents must be tittered on known

positive and negative cell populations prior to use in actual experiments.

2) Controls that are necessary:

a. Negative (no stain whatsoever)

b. or Negative (secondary reagent added only or similarly labeled non-specific

primary antibody, ideally of the same isotype as the specific antibody you will be

using)

c. Negatives and Positives for each different primary antibody, each different animal,

each different strain or species, and each different cell population used in your

experiment, when possible.

3) For two and three color analysis compensation controls must be run to compensate for

spectral overlap. These consist of one sample each stained with each fluorescent reagent

separately and a control containing both colors; e.g. if two-color analysis is performed with

FITC and phycoerythrin then samples stained with FITC alone and PE alone and a sample

certain to be positive for both colors should be run.

https://www.google.ps/?gws_rd=cr,ssl&ei=wXiAVse1BseEUeXVvIgG#q=protocol+for+FITC+staining+for+Fixed+cells

Hoping this will be helpful,

Rafik

Dear Anteneh,

The following is a general protocol:

GENERAL CELL STAINING PROTOCOL FOR FLOW CYTOMETRY

1) Except for cells grown in culture, cells obtained directly from tissues must first be resolved to

a single cell suspension by means of mechanical dissociation (mincing, grinding between the

ends of two frosted glass slides, dounce homogenization, steel mesh sieving, passage through

a fine-bore syringe tip etc.) or enzymatic digestion (collagenase, DNAse, neutral protease

disruption of stromal cells or pepsin digest of solid tissue). Mechanical dissociation of

spleen and thymus is effective in releasing lymphocytes but is often damaging towards

larger, blastic cells and usually leaves dendritic and stromal cells behind in the debris. These

cells are obtained through gentle enzymatic digestion.

Prior to staining, cells must then be purified by at least one of several procedures to enable

analysis on the flow cytometer without damage to the flow cell and permit staining without

excessive background. This will invariably include some combination of density gradient

centrifugation, positive or negative selection procedure (panning, complement-mediated cell

lysis) straining through nylon-mesh and/or red-cell lysis where necessary. (Prior to the BD

FACScan, earlier instruments mandated nylon mesh screening s the smaller flow cells on

these machines could easily become clogged by clumped or aggregated cells and often this

could take months and hundreds of dollars to repair!)

2) Wash purified cells 1X in staining buffer. (A suitable buffer will be isotonic and buffered to

neutrality, will cushion the cells against damage during centrifugation, block non-specific

staining, prevent capping of bound antibody and will block Fc receptor binding.)

e.g. formulations:

.05 M Tris buffered Saline pH 7.4 or: Phospate buffered Saline

1% normal sera of the animal for which 2% Bovine Serum Albumin

the primary antibody is derived or 0.1mg/ml 0.1% Azide

Ig of such animal. (will not block Fc receptor binding)

2% Fetal Calf Serum

0.1% Azide

3) Adjust concentration of cell suspension to at least 5 x 10

6 cells/ml. Cells should be greater

than 90% viable as determined by trypan blue exclusion. Determine # of samples necessary

for experiment and aliquot (1 x 10

5 – 1 x 10

6 cells/sample, preferably the higher value if

possible) to U bottom microtiter well or plastic centrifuge tube.

4a) For staining in microtiter plates. Spin plate for 3 minutes at 100 x g (usually 600 rpm) at

8o

C. Flick plate or remove fluid from wells by suction and immediately resuspend pellet in

100ul of 1st reagent, appropriately diluted to previously determined titration point in staining

buffer (primary antibody directly labeled (e.g. with FITC) if performing direct-labeling,

primary antibody biotinylated or unlabeled if performing indirect staining.) Incubate 30’ at

4o

C. (in the dark if fluorescent labeled)

5a) Wash 3X. (Spin, flick, resuspend in S.B.) After last wash, flick and resuspend in 100ul of

secondary reagent (e.g. FITC labeled Goat anti-mouse Ig or Avidin-FITC also pre-titered)

and repeat step 4 if performing indirect staining, otherwise simply resuspend in 100ul of

staining buffer and transfer to Falcon 2052 tube (this is the only tube that fits the FACScan)

containing 3-500ul of staining buffer. Keep samples on ice and perform FACS analysis. If

samples cannot be run immediately fix them in 1% formaldehyde or 3% paraformaldehyde in

PBS for 15 minutes at 4o

C, wash 1X in staining buffer (samples are left in fix by most

researchers but this writer has found a great deal of cell clumping without fixative removal),

store in fridge in the dark for later analysis.

4a) For staining in tubes. Centrifuge at 300 x g (usually 1000 rpm) for 5 minutes at 8o

C.

Carefully aspirate supernatant from cell pellet and resuspend pellet in 100ul of 1st reagent in

staining buffer as above. Incubate and wash as above for direct or indirect procedures.

Important Things to Remember:

1) Both primary and secondary antibodies or avidin reagents must be tittered on known

positive and negative cell populations prior to use in actual experiments.

2) Controls that are necessary:

a. Negative (no stain whatsoever)

b. or Negative (secondary reagent added only or similarly labeled non-specific

primary antibody, ideally of the same isotype as the specific antibody you will be

using)

c. Negatives and Positives for each different primary antibody, each different animal,

each different strain or species, and each different cell population used in your

experiment, when possible.

3) For two and three color analysis compensation controls must be run to compensate for

spectral overlap. These consist of one sample each stained with each fluorescent reagent

separately and a control containing both colors; e.g. if two-color analysis is performed with

FITC and phycoerythrin then samples stained with FITC alone and PE alone and a sample

certain to be positive for both colors should be run.

https://www.google.ps/?gws_rd=cr,ssl&ei=wXiAVse1BseEUeXVvIgG#q=protocol+for+FITC+staining+for+Fixed+cells

Hoping this will be helpful,

Rafik

HI

how can i stain E.coli ( overnight culture) with FITC.

We have tried some protocol but not workin.

Thanks,