P-FPMI-66 - P-FPMI-66

Materials for Cell Isolation

-7.5% EDTA (Ethylene dinitrolotetraacetic acid disodium salt) (EM Science)

-54% Percoll (Amersham/Pharmacia Lot# 289899)

-HyPure Cell Culture Grade Endotoxin Free water (Hyclone Lot # AML17679)

-Goat anti-mouse IgG (H+L) beads (Miltenyi Biotech Lot# 120 000 288)

-Bovine Serum Albumin Fraction V (Sigma A-1595)

-MACS Running Buffer (0.5 % Bovine Serum Albumin, 2mM EDTA in PBSA).

-mouse anti-bovine CD14 (MM61A VMRD Lot # 0994-0499) 1mg/ml

-Aim V (Gibco/BRL) + 2% FBS (JRH Lot# 9L2102) + 50 nM 2BetaME + 2 mM L-glutamine (Gibco/BRL)

-MACS LS Separation Columns (Miltenyi Biotech # 130-042-401)

-Pre-Separation Filters (Miltenyi Biotech # 130-041-407)

-Midi MACS magnets and stand (Miltenyi Biotech)

-Trypan blue (Gibco/BRL 15250-061)

-FACOLA (Sodium Azide 0.03%, Gelatin 0.02%, PBSA pH 7.3)

-2 % formaldehyde (BDH) in FACOLA

Isolation of PBMC from Bovine Whole Blood

1.Collect peripheral blood by venupuncture using an 18 gauge needle attached to a 60 ml syringe filled with 2 ml of 7.5% EDTA. Transfer blood into 50 ml polypropylene centrifuge tubes using sterile technique.

2.Centrifuge 50 ml polypropylene centrifuge tubes at 2500 rpm (1400 x g) for 20 minutes. Turn centrifuge brake off and set temperature to 20°C.

3.Collect the white buffy coat cell layer into a 50 ml centrifuge tube. Bring final volume to 35 ml using room temperature PBSA supplemented with 0.1% EDTA.

4.Layer 35 ml of cell suspension onto 15 ml of 54 % isotonic Percoll in 50 ml polypropylene tubes.

5.Centrifuge for 20 minutes at 3000 rpm (2000 X g) at 20°C with centrifuge brake turned off.

6.Collect the cells at the interface between Percoll and PBSA in new 50 ml polypropylene centrifuge tube(s).

7.Wash cells once in ice cold PBSA supplemented with 0.1% EDTA, centrifuging for 8 minutes at 1200 rpm (325 X g) at 4°C with centrifuge brake turned on.

8.Repeat step 7 but centrifuge for 8 minutes at 800 rpm (150 X g). Then count cells using a hemacytometer. Assess cell viability using the trypan blue exclusion method.

9.Wash cells a third time in 40 ml Macs Running Buffer centrifuging for 10 minutes at 1400 rpm (400 X g).

10.Resuspend PBMC to a cell density of 108 per ml in Macs Running Buffer.

Primary Labelling of PBMC with Mouse Anti-Bovine CD14

1.Mix 3 ml of MACS Running buffer with 16 µl of mouse anti-bovine CD14 monoclonal antibody (MM61A, 1mg/ml) and 3 ml of PBMC in a new 50 ml centrifuge tube.

2.Mix several times during a 15 minute refrigeration period. Do not keep on ice as this slows down antibody binding.

3.Wash cells by filling tubes with 40 ml cold MACS Running Buffer and centrifuging for 10 minutes at 1400 rpm (400 x g).

4.Discard supernatant and repeat wash as described in step 3.

5.Count and assess cell viability.

Secondary Labelling of PBMC with of Goat Anti-Mouse IgG Magnetic Beads

1.Add 20 nl goat anti-mouse IgG magnetic beads per 107 cells and 80 nl MACS Running Buffer per 107 cells. Make sure you have a single cell suspension.

Mix and refrigerate for 15 minutes.

2.Repeat steps 3, 4 and 5.

3.Collect a sample of these stained cells for flow cytometry analysis.

Separation Gravity Macs

1.Resuspend cell pellet 108 per ml in Macs buffer. Cell pellets less than 108 per ml are resuspended in a 1 ml volume of Macs buffer.

2.Pass cell suspension through Miltenyi Biotech Pre-Separation Filters (130-041-407). Place Pre-Separation filter on a 15 ml polypropylene tube. Wet the filter by vigorously pipetting 500 µl of running buffer through it. Place Pre-Separation Filter unit on top of a pre-wetted LS Midi column (to pre-wet a midi column, mount the column onto the magnet and pass 3 ml of Macs Running Buffer through it). Pipette cell suspension through the Pre-Separation filter and into the LS Midi separation column. Make sure you have a 15 ml conical centrifuge in place to collect the non adherent cells.

3.Once the final drop has passed through the column, wash cells by passing 3 ml of Macs buffer through the column.

4.Repeat step 12 for a total of 3 washes.

5.Remove the column from the magnet and place into a sterile 15 ml conical centrifuge tube. Add 5 ml of Macs buffer and push through the column using the plunger provided with the column.

6.Pellet adherent and non adherent cell fractions by centrifuging for 10 minutes at 1000 rpm and 4oC.

7.Repeat steps 10-15 except pass only the adherent cell fraction through the second column.

8.Count and resuspend adherent and non adherent fractions to desired cell density in the appropriate culture medium

Flow Cytometry Analysis of PBMC, Adherent, and Non-Adherent Cell Fractions

Stain both positive and negative fractions using the panel of monoclonals listed below:

Mouse Anti-Bovine Monoclonal Antibodies:

-MM1A ((CD3) 1/400 of 1 mg/ml (VMRD)

-MM61A ((CD14) 1/200 of 1 mg/ml (VMRD)

-BAQ15A ((CD21) 1/300 of 1 mg/ml (VMRD)

-ILA11 ((CD4)1/300 of 1 mg/ml (VMRD)

-BAQ111A ((CD8) 1/300 of 1 mg/ml (VMRD)

-CD172a 1/400 ((Serotech #MCA2041S)

Purified Mouse Isotype Control Antibodies:

-IgG1 (Caltag MG100)

-IgG2a (Caltag MG2a00)

-IgG2b (Caltag MG2b00)

-IgM (Caltag MGM00)

Goat Anti-Mouse Secondary Fluorochromes:

-IgG1 FITC (Southern Biotech #1070-02)

-IgG2b PE (Caltag M32404)

-IgM PE (Caltag M31504)

-IgG2a PE (Caltag M32204)

Time Course For Resting Monocytes:

A)Wash newly isolated monocytes once in 15 ml PBSA (ie. No EDTA).

Centrifuge the 15 ml centrifuge tube for 10 minutes at 1000 rpm at 10oC.

Resuspend monocytes 107 per ml in culture medium (Aim V + 2% FBS heat

inactivated + 50 M 2Me, + 2 mM L-glutamine). Add 0.5 ml of cells (5

million CD14+) to each well on a 12 well Costar Tissue Culture plate. Add 2.5 ml of culture medium to each well to make the final volume 3 ml.

B) Rest the cells for 20h 37oC with 5% CO2 and 96% humidity in air.

C) Harvest a portion of the cells and call them time zero. Harvest by washing cells as follows: remove culture supernatant from each well into a clean 15 ml Corning centrifuge tube. Wash cell monolayer 2x with 1 ml PBSA (no EDTA). Transfer the wash solution from each well into its own 15 ml centrifuge tube. Add 500 l of Trizol (Sigma)

to each well prior to centrifugation of the wash supernatant. Centrifuge the tubes containing wash solution for 1400 rpm for 5 minutes at 4oC.

Discard supernatant and resuspend cells in 100 l of Trizol (Sigma). Add the Trizol resuspension to the corresponding well from which the cells originated. Seal plate with parafilm and freeze at -20 oC.

Experiment E-FPMI-2