Isolation of human peripheral blood mononuclear cells (PBMC) and CD4+ T cells
1| Transfer blood in heparin vacutainer tubes into 50 ml BLUE MAXTM BD Falcon tube
2| Carefully layer 30 ml diluted blood over 12ml of Isolymph (do not allow layers to mix)
3| Centrifuge for 30 min (1,800 rpm) at room temperature.
4| Harvest buffy coat layer (whitish inter-phase layer containing PBMC) (~2-5 ml)
5| Transferred to fresh 50 ml Falcon tube and bring to 50 ml with PBS
6| Wash twice in PBS by centrifugation for 10 min in 1,200 rpm at 4 °C
7| Suspend pellet in 5 ml cold complete medium and count cells in hemacytometer.
8| 15-20 ml of buffy coat yields >50×106 buffy coat cells
9| Isolate CD4+ T cells from buffy coat with Rosette Sep kit as suggested by manufacturer.
Expansion of Th17 cells in human PBMC by IL-2
Cell cultures and ELISA
1| Prepare fresh culture medium containing requisite antibiotics and serum
2| Re-suspend PBMC or purified CD4+ T cells (1 X 106 per ml) in presence or absence of IL-2 (100 u/ml), TGF-β1, IL-6 or IL-23 at 10 ng/ml or IFN-γ at 100 U/ ml.
3| Culture cells in 24 well plate (2 ml per well) at 37 °C for 4 days in 5% CO2
4| Harvest culture supernatants (0.5 ml/well) for ELISA.
5| Multiplex ELISA was performed using Pierce Search Light Technology (Pierce Boston Technology, Woburn, MA).
6| Harvest cells by pipetting gently up and down with 2 ml Serological pipette
7| Transfer to 15 ml tubes and centrifuge at 1200 RPM (Sorvall Legend RT) for 10 min.
Intracellular cytokine staining assay
1| Activate PBMC or CD4+ T cells in the presence or absence of IL-2 for 4 days
2| Stimulate cultures with PMA (20 ng/ml) and Ionomycin (1 mm) for 4 h
3| Terminate the reaction by addition of Golgistop to cultures for 1 additional hour
4| Transfer cells into 15 ml of SARSTEDT tubes and wash cells twice in FACS buffer
5| Suspend cells in FACS buffer (1-2×106/ml)
6| Transfer 1 ml aliquots (1-2×106) into separate 5 ml Falcon tubes
7| Centrifuge for 1,200 rpm for 10 min.
8| Perform intracellular staining using BD Cytofix/Cytoperm kit as suggested by manufacturer
9| Subject stained cells to FACS analysis immediately or within 48 h (keep cells at 4 °C). In our lab, data collection was with FACSCalibur and analyzed using FLOWJO analysis software.
RT-PCR and Quantitative RT-PCR (qRT-PCR)
RNA Isolation using TRIZOL Reagent
1| Homogenize samples in 1 ml of TRIZoL reagent per 50-100mg of tissue (<15 ml)
2| Transfer to Nalgene tubes and incubate for 5 minutes at room temperature.
3| Add 0.25 ml of chloroform per 1 ml of TRIZoL reagent.
4| Shake vigorously by hand or vortex for 15 seconds.
5| Incubate at room temperature for 2-3 minutes.
6| Centrifuge at 12,000 g (about 9,000 rpm on SV600) for 15 min at 4 °C.
7| Transfer aqueous phase to fresh Nalgene tubes.
8| Add equal volume of isopropyl alcohol.
9| Mix well and incubate samples at room temperature for 10 minutes.
10| Centrifuge at 12,000 g for 10 minutes at 2-8 °C.
11| Discard supernatant and add 1 ml of 75% ETOH to pellet.
12| Using a 2 ml pipette, scrape the RNA pellet away from tube into the ETOH.
13| Transfer ETOH/RNA mixture to fresh 1.5 ml eppendorf tube.
14| Rinse nalgene tube with more 75% ETOH and transfer to the eppendorf tube.
15| Bring the final volume of RNA/ETOH sample to 1.5 ml with 75% ETOH
16| Centrifuge at max speed in refrigerated microfuge for 10 minute
17| Discard supernatant and add 500 µl 75% ETOH
18| Centrifuge at 10,000 rpm in refrigerated microfuge for 5 minute
19| Pulse the sample and using P100 pipette carefully remove residual ETOH.
20| Air-dry RNA pellet for 15 minutes.
21| Redissolve RNA in DEPC treated water and store in aliquots at -70 °C.
22| Determine RNA concentration using a spectrophotometer.
DNAse Digestion of RNA Samples
1| To 1.5 ml microcentrifuge tube add the following (all RNA grade):
RNA =X µl
10X Buffer =50 µl
DNAse I =1 µl
DEPC Water ==>500µl
Note: Can scale down to 50 µl
2| Digest for 20 minutes at 37 °C.
3| Add 30 µl 20 mM EDTA (RNA grade) and heat at 95 °C for 5 mins.
4| Phenol/chloroform extraction (1x): add 500 µl (Phenol/chloroform
5| Vortex (1 minute) and centrifuge for 5 minutes at 14,000 rpm (RT).
6| Transfer aqueous phase (top) to another RNAse-free 1.5 ml tube.
7| Chloroform extraction: Add 400 µl of chloroform (with isoamyl alcohol)
8| Vortex (1 minute) and centrifuge for 5 minutes at 14,000 rpm (RT).
9| Transfer aqueous phase (top) to another RNAse-free 1.5 ml tube.
10| Determine the volume of sample
11| Precipitate by adding 1/9 volume of 3 M NaOAc, pH 5.2.
12| Add 2 volumes cold absolute (95-100%) alcohol
13| Incubate on dry ice (or -70 °C) for 15 minutes.
14| Centrifuge at 4 °C, 14,000 rpm for 15 minutes.
15| Remove supernatant and add 500 µl of ice-cold 75% EtOH.
16| Incubate for 5 minutes at RT.
17| Centrifuge at 4 °C, 14,000 rpm for 5 minutes.
18| Remove supernatant and add 500 µl of ice-cold 75% EtOH.
19| Incubate for 5 minutes at RT.
20| Centrifuge at 4 °C, 14,000 rpm for 5 minutes.
21| Remove supernatant
22| Spin for 30 seconds
23| Remove supernatant with 100 µl pipetteman
24| Air dry for 15 minutes.
25| Suspend in DEPC water.
First Strand cDNA Synthesis
Use only DEPC-treated H2O, RNase-free tubes, and ART tips
1| Wipe down lab bench and pipetteman with 10% H2O2 and change gloves frequently
2| Mix all components briefly and spin down.
3| Preset heat blocks or thermocycler to 37 °C, 50 °C and 70 °C, respectively
4| Into a 0.5 ml microcentrifuge tube add 27.5 µl RNA (1-10 µg)
5| Add 2.5 µl Oligo(dT) to each tube
6| Incubate mixture at 70 °C for 10 minutes
7| Quench on ice for 1 minute and leave on ice
8| Add the following reagents in the following order:
5 X First Strand buffer 10 µl
a. M DTT 5.0 µl
10 mM dNTP 2.5 µl
RNAse out 1.0 µl
9| Incubate for 2 minutes at 50 °C
10| Add 2.5 µl Superscript III (Invitrogen; Catalog # 18080-093)
11| Mix gently by tapping tubes, pulse
12| Incubate at 50 °C for 60 minutes
Note: Up to this point, be extremely careful to prevent RNase contamination. For preparation of more than 5 RNA samples, Superscript can be added together with the other reagents
13| Incubate at 70 °C for 15 minutes to terminate reaction
14| Incubate tubes at 37 °C for 2 minutes
15| Add 1.0 µl (3.5 units) of RNase H, mix gently by tapping tubes
16| Incubate at 37 °C for 30 minutes
17| Then at 95 °C for 5 minutes
18| Pulse samples by brief centrifugation
19| Add 6 µl of 4 M LiCl to each tube, vortex and pulse
20| Add 150 µl of cold (-20°C) absolute EtOH and vortex
21| Freeze in dry ice for 30 minutes
22| Spin at 14,000 rpm for 15 minutes and remove supernatant
23| Add 150 µl of cold 75% EtOH
24| Spin for 15 minutes as above and remove supernatant
25| Air-Dry for 15 minutes
26| Suspend pellet in 50 µl of DEPC-treated H2O
27| Incubate at 70 °C, 10 minutes: to completely dissolve the pellet
28| Save 0.5-1.0 µl for cDNA quantification
29| Store at -20 °C
Note: Verify that all RNA preparations are of high quality by spectrophotometry (optical density at 260 Angstrom: >1.9) and
30| Verify integrity of the RNA by Formamide electrophoresis using 1.5-2.0 µg RNA (use mini-gel apparatus). Examine intensity of 18S and 28S ribosomal RNA.
31| For RT-PCR, incubate samples at 95 °C for 10 min to activate the AmpliTaq Gold and carry out amplification for 30-35 cycles of 30 s each at 95 °C, 60 °C, and 72 °C. Follow this by a final 10-min extension at 72 °C. PCR primers used span at least an intron.
32| Perform real-time 5’-nuclease fluorogenic RT-PCR analysis on an ABI 7500 real-time PCR System using Taqman primer/probe sets (ABI) and PCR parameters as recommended for TaqMan Universal PCR master mix kit (PE Biosystems). Calculate relative levels of gene expression by extrapolation from standard curves generated using purified human IL-17 cDNA fragments. Standard curves from our lab showed excellent linearity, indicating precise quantitative relationship between cDNA copy number and fluorescence intensity within the dynamic range of the assay.
33| Normalise the mRNA expression levels to the levels of ACTB (encoding β-actin) and/or GAPDH housekeeping genes