Dear Ranin,

We use, 1x PBS (or HBSS) with 2% Heat inactivated FBS and sodium Azide at 0.1% the pH  7 to 8, for our FACS FLOW buffer.

Best,

Charles

Hello my dear 

Preparation of peripheral blood mononuclear cells and

phenotyping by multi-colour flow cytometry 2.1

Blood was drawn from healthy volunteers following informed consent (see

Appendix for relevant forms) into EDTA Vacutainer® tubes (Becton Dickinson

(BD) Biosciences, San Jose, CA, USA). A total of 3 ml of anticoagulated blood

were layered over 3 ml of Ficoll Paque® density gradient medium (GE

Healthcare, Little Chalfont, UK) in a 15 ml falcon tube (Sarstedt, North RhineWestphalia, Nümbrecht, Germany) and the tubes were centrifuged at 400 RCF

for 22 minutes at 4°C with full acceleration and no braking. Afterwards, a thin

cloudy layer containing PBMCs (“buffy coat”) was carefully removed using a

plastic Pasteur pipette (Sarstedt). This was transferred to a 15 ml Falcon® tube

and the volume was made up to 10 ml with FACs buffer [2% foetal calf serum

(FCS) (Lonza, Basel, Switzerland) PBS, 0.05% NaN3 (Sigma-Aldrich, , St. Louis,

MO, USA)]. The cell suspensions were pelleted by centrifugation at 400 RCF for

5 minutes at 4°C with full brake and acceleration. Supernatants were discarded

and cell pellets were re-suspended in 5 ml of FACs buffer and washed again

using the same procedure. The final cell pellets were re-suspended in 1 ml of

FACs buffer and counted using a haemocytometer. Aliquots of 500,000 cells

each were transferred to 3 ml polystyrene FACs tubes (Sarstedt) and the final

volumes were made up to 100μl with FACs buffer. Cells were stained with

combinations of fluorochrome-conjugated antibodies directed against various

cell surface markers and expression levels were subsequently expressed as

mean or median fluorescent intensity (MFI) as appropriate. Flow cytometric

analyses were carried out in the NUI Galway Flow Cytometry Core Facility using

a FACSCanto A or FACSCanto II cytometer (BD Biosciences). Compensation and

gate settings were generated using compensation beads (Life Technologies,

Carlsbad, CA, USA) and Flow Minus One (FMO) controls. Data files were

analysed using FlowJo v.7 software (Tree Star Inc. Ashland, OR, USA).

May-Grunwald and Giemsa staining of monocyte subsets 2.2

Individual monocytes subsets were isolated using fluorescence activated cell

sorting (FACS) (see Section 2.4). Between 10,000 and 20,000 cells were resuspended in 200 μl of FACs buffer and spun onto glass microscope slides

(VWR, Radnor, PA, USA) at 200 rpm for 5 minutes using a Shandan Cytospin 3,

(Thermo Fisher Scientific, Waltham, MA, USA). The slides were air dried for 10

minutes at room temperature before being fixed for 5 minutes in 100%

methanol (Sigma-Aldrich). The slides were air dried for 2 minutes at room

temperature then were stained for 5 minutes in undiluted May-Grunwald stain

(Sigma-Aldrich). Next, the slides were washed three times for 2 minutes each

in phosphate buffered saline (PBS), pH 7.2, then stained in Giemsa stain

(Sigma-Aldrich) (diluted 1:20 with deionized water) for 10 minutes. Finally, the

slides were washed 3 times in deionized water for 20 seconds per wash and

were air dried for 10 minutes with excess stain wiped from the back of the

slide using tissues soaked in industrial methylated spirits (Sigma-Aldrich).

Bright field images were immediately captured on an Olympus BX43

microscope (Olympus Corporation, Shinjuku, Tokyo, Japan).

Hello Ranin.

I could try fix with transfix. A solution to keep the cell viability for flow cytometry.

Please look the file

Best,