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Isolation of Mononuclear Cells from solid tumorsNatascha 12.12.09 modified after: T helper 17 cells promote cytotoxic T cell activation in tumor immunity. Martin-Orozco N, Muranski P, Chung Y, Yang XO, Yamazaki T, Lu S, Hwu P, Restifo NP, Overwijk WW, Dong C. Immunity. 2009 Nov 20;31(5):787-98; and (Vremec et al., 2000).
Exp:… ……… /………………..……EP……
Need: camera, scissors and pinzette Petri-dishes, PBS with 2% FCS Ruler, Nunc-vial, dry-ice
Isolate draining (inguinal) and spleen per mouse Take lungs in 2ml 10% Formalin, take blood for serum, take prostate/tumor (some for -80°C)
different FACS analysis due to multi-color staining on Fortessa, spleen - Calibur
Mice: Body weight of the mice in RN Tumor weight in Lab
mouse weight +Seminal V -prostate 93
1. Measure tumor volume- prepare tumor from killed mice- kill mice individually take a picture with a ruler! +/- Seminal vesicles -> all together
2. Cell isolation from tumor place the tumor in a 6-well plate from the petri-dish containing ice-cold PBS + 2% FCS Cut the tumor into small pieces (razor blade or small knifes) in a 6 well plate digest with 1 mg/ml collagenase D for 45 min at 37°C (5 ml / tissue) number of mice: 1 5 ml For 10ml: take 8.55 ml PBS 4,1 ml + 100µl Collagenase (250mg/ml) / (100mg/ml) 50µl / 125µl + 100µl DNase (100mg/ml) / (20mg/ml) 50µl / 250µl + 1ml MgCl (for DNase activity 25mM) 500µl + 200µl Serum 100µl pre-warm at 37°C
5 min at 37°C with 0.01 EDTA for prevention of DC-T cell aggregates + 100µl EDTA (0.5M) per 5ml put through a 70 µm cell strainer placed in a 6 well dish (back of 1 ml syringe plunger) Collect the single cell suspension into a 15 ml tube on ice Wash the cell-strainer with PBS and add to the tube until the solution is clear. Take small aliquot (250µl) of digested tumor cells for (cell counting) surface staining (CD4 PE, CD8 APC, CD45 FITC, DX-5 PE-Cy7). Centrifuge for 8-10 min at 1400rpm end: 1 TU samples
Meanwhile isolate draining LN and spleens with cell strainer
Prepare the Percoll 30% and 70% in PBS (+Serum) (Sigma P1644-500ML)! 20ml 30% Percoll: 6ml +14ml PBS: number of mice: 5ml x …1.. = …5……..ml 10ml 70% Percoll: 7ml + 3ml PBS: number of mice: 2,5ml x …1.. = …2,5……..ml
· Cells are resuspended in 30% Percoll: 5ml o Turn the pipette boy to S (slow) o Overlay very slowly with the lower (and lighter) 30% Percoll solution containing the cells onto the already provided 70% Percoll 2,5ml o Centrifuge (1400rpm, 20min, RT , NO BRAKE) o Remove the surface – tumor cell debris and fat with 5ml pipette or 1ml Tip o Carefully suck off the interphase with mononuclear cells by using the pipette boy and get rid of Percoll contamination by washing · Transfer into 15ml tubes, wash two times with PBS (+2%FCS) · Centrifuge (1400rpm, 7min, 4°C)
Split cells: 1/3 for surface and 2/3 for IC (1/3 eBioscience [IC C], 1/3 Biolegend [IC D]) Start with restimulation!! (IC D)
Take 1/3 surface and split again – use 1/3 for Surface B (Calibur) and other 1/3 for Surface A (Fortessa), 1/3 for Surface M (Calibur) 2/3 of cells for IC (1/3 has to be restimulated for 4 hours – proceed on page 5), 1/3 for eBio TF Fortessa staining
Tumor infiltrates isolation modified for colon(Mario Kuttke) Materials:
Reagents:
o DNase I (Worthington Biochemicals LS 002139) 5 mg o Collagenase P ( Roche 11249002001) 12.5mg o Collagenase/Dispase (Roche 11097113001) 12.5mg o RPMI to 5ml o Filter sterilize 0.22µm
· DNASE SOLUTION (1ml per tumor) o Can be aliquoted and stored at –20oC. Make fresh solution is best! o DNase I (Worthington Biochemicals LS 002139) 0.5 mg o RPMI to 1 ml o filter sterilize on 0.45 micron filter · Percoll gradient (2x if you want to isolate IELs as well) · Mix 9 vol Percoll with 1 vol PBS 10x = Percoll 100 4ml (3,6ml Percoll +0,4 ml 10xPBS) · Make up Percoll 35 (35% Percoll 100 + 65% PBS) 4ml (1,4ml+2,6ml) · Make up Percoll 60 (60% Percoll 100 + 40% PBS) 4ml (2,4ml+1,6ml) · Prepare your gradients as follows: · Add 4 ml Percoll 35 to a 15 ml tube and underlay with 4ml Percoll 60
Tumor associated macrophage isolation PROCEDURE:
a. Aspirate the cells that are located between the 5ml suspension medium and 35% percoll. Usually this cell layer comes easily as one sheet. b. With a transfer plastic pipette, harvest the tumor cells / macrophages that settled on the 60% Percoll. Try to remove all the cells, put them in a 50 ml tube, fill up with PBS. Spin 5 minutes, 300x g, +4oC, BRAKES ON.
B16-OVA tumor induction(Julia Pisoni) http://www.lgcstandards-atcc.org/products/all/CRL6475.aspx?geo_country=at B16-OVA Tumormodell zur Ermittlung der Compound-Wirkung auf die Anti-Tumor Immunität: WT-Mäuse werden, unter oraler Verabreichung der Compounds, mit B16 Zellen orthotopisch subcutan in die Flanke inokuliert. Dabei werden die Mäuse mit Isofluran betäubt und 1x106 B16-Zellen werden in 200µl einer 1:2 PBS Matrigel Mischung mit einer Insulin Spritze (23 Gauge Kanüle) injiziert. Nach dem sichtbaren Anwachsen des Tumors (Tag 6) werden die Tiere 2x mit OVA-Antigen vakziniert (Tag 6, Tag 13) und das Wachstum bzw. die Abstossung des Tumors wird entsprechend dokumentiert. Der Versuch wird am Tag 21 beendet bzw. werden Mäuse aus ethischen Gründen vorher euthanasiert wenn der Tumor zu einer schweren Beeinträchtigung führt. Am Versuchsende wird der Tumor für die Histologie aufbereitet sowie die T-Zellen der Tumor Draining Lymphnodes mittels Durchflusszytometrie analysiert.
Exp.: 15.10.2013 è Thaw at least 1- 1,5 week prior to the injection and split at least two times
1x 105 B16-OVA cells injected subcutaneously into
de-haired (w) or shaved (m) left flanks of 1 *105 in 100 µl
How many mice: 1x: 100.000: ……8…mice… need …8*105 ……… cells in ……800µl EXTRA: 3x: 1.000.000: ……24………… need …2,4*106 ……… cells in ……2400µl
1. Injection of B16- melanoma cells: B16 –Ova- Melanoma cells: (modified after Christina 07.01.10)
Growth: DMEM +++: Medium β-mercaptoethanol. (500ml + 2µl) Split every second day (except weekend) 1:4 in relation to the area Use cell culture dishes- not petri dishes- or cell culture flasks to grow in Cells must be in their exponential growing phase for optimal results DMEM + · G418- BC (30.000U/ml) Biochrom: 80µl mikroliter pro 10ml. · 10% FCS, · Glutamat · PenStrep
Harvesting: Remove DMEM medium Wash with PBS (2x) Add 2-3 ml Trypsin (+EDTA Nr……) per flask (4 flasks for 18 mice) Incubate for 3-5 min at 37°C Add DMEM+++ medium (serum stops trypsin) and wash 2 times (12000rpm 7min) (Zellen kleben stark, gut resuspendieren)
Freezing 5x106 cells/ml 1:10 in DMSO FCS in the Mister cooler -80°C Defrost fast – put into 37°, wash well with DMEM to get rid of the DMSO and put into a flask.
Injection: Be careful not smaller than 0,4mm needle (27 gauge) 1x 105cells in 100µl PBS mix gently every time you take out an aliquot- no air bubbles! Use matrigel if tumor is subjected to IHC staining!
I.p or (depending on tumor model) i.v. inject the mice- that have been below the red light for at least 15min.
measure tumor growth every 2nd day with a caliper
StainingsImmunohistochemistry Stainings (IHC)Arginase 1 IHC Staining- 5-10 min 60°C - Rehydrate (10min Xylen, 30sec. 100%, 96%, 80%, 70%, 50%), A. dest. - Antigenretrieval steamer pH 6, 1h - Let it cool off, then wash 3x in 1XPBST - Block 10min in 3%H2O2 (in PBS) - Wash 3x in 1XPBST - 10min Avidin block - Wash 3x in 1XPBST - 10min. Biotin block - Wash 3x in 1XPBST - Surround tissue with DAKO pen - 7min. Superblock (2 drops) - Wash 3x in 1XPBST - 1h mouseblock (2 drops) - Wash 3x in 1XPBST - 1° ab (Arg 1) 1h at RT / on at 4°C (1:500 in PBS 1% goat serum) - Wash 3x in 1XPBST - 10min. Biotinylated ab - Wash 3x in 1XPBS (NO TWEEN) - 10.min Streptav.-HRP - Wash 3x in 1XPBS (NO TWEEN) - AEC ( 20µl substrate +1000µl buffer) - stop in tab water - Wash in aqua dest. - Hemalaun - stop in tab water - mount with Aquatex
sc-20150 Arginase I (H-52) rabbit polyclonal
IgG Santa Cruz FoxP3 / IL-17 double IHC Staining- 30min-1h 60°C - Rehydrate (3x10min Xylen, 30sec. 100%, 96%, 80%, 70%, 50%), A. dest. - Antigenretrieval steamer pH 9, 1h - Let it cool off, then wash 3x in 1XPBS - Block 10min in 3%H2O2 (in PBS) - Wash 3x in 1XPBS - 10min Avidin block - Wash 3x in 1XPBS - 10min. Biotin block - Wash 3x in 1XPBS - 7min. superblock - Wash 3x in 1XPBS - 1h mouseblock - Wash 3x in 1XPBS - 1° ab (FoxP3) 1h at RT (1:200 in PBS 1% BSA) - Wash 3x in 1XPBS - 10min. Biotinylated ab - Wash 3x in 1XPBS - 10.min Streptav.-HRP - Wash 3x in 1XPBS - AEC - Wash in aqua dest., then 1XPBS - Block 1h in 5% goat serum in PBS - 2° ab (IL-17) 1h at RT or overnight (1:200 in PBS 1% BSA) - Wash 3x in 1XPBS - 30min. anti-rabbit biotinylated ab 1:100 - Wash 3x in 1XPBS - 10.min Streptav.-HRP (ID labs) - Wash 3x in 1XPBS - AEC(20µl substrate + 1000µl Buffer)/ histogreen develop, stop in tap water - Wash in Aqua dest. - Hemalaun 1:5, 30s, stop in tap water - Aquatex
Foxp3(F-9) sc-166212 mouse mono IL-17 Abcam ab91649 All washing steps in PBS-T (0.1%) until biotinyl. Ab, then only PBS! Rabbit polyclonal: anti-rabbit biotinylated ab 1:100
DAB/Metal Concentrate Thermo scientific # 34065 (=new cat. Number) 1:10
Histogreen Linaris #E109
CD3 IHC Staining- 5-10 min 60°C - Rehydrate (10min Xylen, 30sec. 100%, 96%, 80%, 70%, 50%), A. dest. - Antigenretrieval steamer pH 6, 1h - Let it cool off, then wash 3x in 1XPBST - Block 10min in 3%H2O2 (in PBS) - Wash 3x in 1XPBST - 10min Avidin block - Wash 3x in 1XPBST - 10min. Biotin block - Wash 3x in 1XPBST - Surround tissue with DAKO pen - 7min. Superblock - Wash 3x in 1XPBST - 1h mouseblock - Wash 3x in 1XPBST - 1° ab (CD3) 1h at RT / o n at 4°C (1:150-300 in PBS 1% goat serum) - Wash 3x in 1XPBST - 10min. Biotinylated ab - Wash 3x in 1XPBS (NO TWEEN) - 10.min Streptav.-HRP - Wash 3x in 1XPBS (NO TWEEN) - AEC ( 20µl substrate +1000µl buffer) - stop in tab water - Wash in aqua dest. - Hemalaun - stop in tab water - mount with Aquatex
anti-CD3 (Early T Cell Marker), Clone: SP7, Thermo Scientific
CD8a IHC Staining- 30min-1h 60°C - Rehydrate (3x10min Xylen, 30sec. 100%, 96%, 80%, 70%, 50%), A. dest. - Antigenretrieval steamer pH9, 1h - Let it cool off, then wash 3x in 1XPBST - Block 10min in 3%H2O2 (in PBS) - Wash 3x in 1XPBST - 10min Avidin block - Wash 3x in 1XPBST - 10min. Biotin block - Wash 3x in 1XPBST - 7min. superblock - Wash 3x in 1XPBST - 1h mouseblock - Wash 3x in 1XPBST - 1° ab 1h at RT (1:100 in PBS 1% BSA) - Wash 3x in 1XPBST - 10min. Biotinylated ab - Wash 3x in 1XPBS - 10.min Streptav.-HRP - Wash 3x in 1XPBS - AEC (20µl substrate + 1000µl Buffer), develop, stop in tap water - Wash in aqua dest. - Hemalaun 1:5, 30s, stop in tap water - Aquatex
All washing steps in PBS-T (0.1%) until biotinyl. Ab, then only PBS!
Rat-anti-mouse CD8a (53-6.7) Biolegend cat# 100701 Stored at 4°C
CD163 IHC Staining- 30min-1h 60°C - Rehydrate (3x10min Xylen, 30s. 100%, 96%, 80%, 70%, 50%), A. dest. - Antigenretrieval steamer pH9, 1h - Let it cool off, then wash 3x in 1XPBS - Block 10min in 3%H2O2 (in PBS) - Wash 3x in 1XPBS - 10min Avidin block - Wash 3x in 1XPBS - 10min. Biotin block - Wash 3x in 1XPBS - 7min. superblock - Wash 3x in 1XPBS - 1h mouseblock - Wash 3x in 1XPBS - 1° ab 1h at RT (1:200 in PBS 1% BSA) - Wash 3x - 10min. Biotinylated ab - Wash - 10.min Streptav.-HRP - Wash - AEC (20µl substrate + 1000µl Buffer), develop, stop in tap water - Wash in aqua dest. - Hemalaun 1:5, 30s, stop in tap water - Aquatex
CD163 sc33560 (M-93) Rabbit polyclonal IgG
All washing steps in PBS-T (0.1%) until biotinyl. Ab, then only PBS!
ERG IHC Staining- 5-10 min 60°C - Rehydrate (10min Xylen, 30sec. 100%, 96%, 80%, 70%, 50%), A. dest. - Antigenretrieval steamer pH 9, 1h - Let it cool off, then wash 3x in 1XPBST - Block 10min in 3%H2O2 (in PBS) - Wash 3x in 1XPBST - 10min Avidin block - Wash 3x in 1XPBST - 10min. Biotin block - Wash 3x in 1XPBST - Surround tissue with DAKO pen - 7min. Superblock - Wash 3x in 1XPBST - 1h mouseblock - Wash 3x in 1XPBST - 1° ab (ERG EP111) 1h at RT / on (1:50eigentlich) in DAKO antibody diluent) - Wash 3x in 1XPBST - 10min. Biotinylated ab - Wash 3x in 1XPBS (NO TWEEN) - 10.min Streptav.-HRP - Wash 3x in 1XPBS (NO TWEEN) - AEC ( 20µl substrate +1000µl buffer) - stop in tab water - Wash in aqua dest. - Hemalaun - stop in tab water - mount with Aquatex
F4/80 IHC Staining- 30min-1h 60°C - Rehydrate (3x10min Xylen, 30sec. 100%, 96%, 80%, 70%, 50%), Aqua dest. - Antigenretrieval microwave pH6 (3min. 800 W, 15 min 290 W) - Let it cool off, then wash 3x in 1XPBS - Block 10min in 3%H2O2 (in PBS) - Wash 3x in 1XPBS - 10min Avidin block - Wash 3x in 1XPBS - 10min. Biotin block - Wash 3x in 1XPBS - 7min. superblock - Wash 3x in 1XPBS - 1h mouseblock - Wash 3x in 1XPBS - 1° ab overnight (1:100) - Wash 3x - 10min. Biotinylated ab - Wash - 10.min Streptav.-HRP - Wash - AEC (20µl substrate + 1000µl Buffer), develop, stop in tap water - Wash in aqua dest. - Hemalaun 1:5, 30s, stop in tap water - Aquatex
1° rat-anti-F4/80 1:100 in 1% goat serum; 150-200μl per slide
F4/80 staining (double staining with P-Stat3)
1° rat-anti-F4/80 1:100 in 1% goat serum; 150-200μl per slide
2° anti polyvalent biotinylated antibody (kit from ID labs contains also super block, mouse block) IDST1007 ? The P-Stat3 staining done by Michi in AKH, with AP, not with HRP system! P-Stat3 (Y705) Rabbit mAb (D3A7) Cell Signaling #9145L
PYStat3 staining
2° anti polyvalent biotinylated antibody (kit from ID labs contains also super block, mouse block) IDSTM003
P-Stat3 (Y705) Rabbit mAb (D3A7) Cell Signaling #9145
F4/80+P-Stat3 for IF
1° Ab: 1° rat-anti-F4/80 1:100 in 1% goat serum; 150-200μl per slide
P-Stat3 (Y705) Rabbit mAb (D3A7) Cell Signaling #9145L
2° Ab: goat anti rat PE (invitrogen A10522) 1:200 goat anti rabbit FITC (invitrogen A11008) 1:200
Total Stat3 H190 (Santa Cruz): 1:80
F4/80+Total Stat3 for IF
1° Ab: 1° rat-anti-F4/80 1:100 in 1% goat serum; 150-200μl per slide
Total Stat3 H190 (Santa Cruz): 1:80 cat.# sc-7179 1:80
2° Ab: goat anti rat PE (invitrogen A10522) 1:200 goat anti rabbit FITC (invitrogen A11008) 1:200
Total Stat3
F4/80+Total Stat3
F4/80 & Total Stat3
- 1h 60°C - Rehydrate (3x10min Xylen, 30sec. 100%, 96%, 80%, 70%, 50%), Aqua dest. 5min. - Antigenretrieval autoclave 20min. pH6 - Let it cool off, then wash 3x in 1XPBS - Block 10min in 3%H2O2 (in PBS) - Wash 3x in 1XPBS - 7min. superblock - Wash 3x in 1XPBS - 1h 5%goat serum in PBS - 1° ab mix overnight 4°C - Wash 3 times in PBS-T - 2nd antibody mix 30min, wash 3 times in PBS-T - Wash 3 times in PBS-T - DAPI (1:50.000) 5min. - Wash 3 times in PBS-T - Dako mounting media
1° Ab: 1° rat-anti-F4/80 1:100 in 1% goat serum; 150-200μl per slide
Total Stat3 H190 (Santa Cruz): 1:80 cat.# sc-7179 1:80
2° Ab: goat anti rat PE (invitrogen A10522) 1:200 this time 1:300
goat anti rabbit FITC (invitrogen A11008) 1:200
Gr1 IHC Staining- 30min-1h 60°C - Rehydrate (3x10min Xylen, 30sec. 100%, 96%, 80%, 70%, 50%), A. dest. - Antigenretrieval steamer pH9, 1h - Let it cool off, then wash 3x in 1XPBS - Block 10min in 3%H2O2 (in PBS) - Wash 3x in 1XPBS - 10min Avidin block - Wash 3x in 1XPBS - 10min. Biotin block - Wash 3x in 1XPBS - 7min. superblock - Wash 3x in 1XPBS - 1h mouseblock - Wash 3x in 1XPBS - 1° ab 1h at RT (1:200 in PBS 1% BSA) - Wash 3x in 1XPBS - 10min. Biotinylated ab - Wash 3x in 1XPBS - 10.min Streptav.-HRP - Wash 3x in 1XPBS - AEC (20µl substrate + 1000µl Buffer), develop, stop in tap water - Wash in aqua dest. - Hemalaun 1:5, 30s, stop in tap water - Aquatex
Gr1 Rat anti mouse Ly6B.2 Serotec MCA771GA 1:200
iNOS mouse IgG BD #610431 1:200
PTEN IHC Staining- 5-10 min 60°C - Rehydrate (5 min Xylen, 2 min. 96%, 96%, 1min 80%, 70% A. dest. - Antigen retrieval (steamer pH 9, 1h) - Let it cool off, then wash 3x in 1XPBST (0,1% TWEEN) - Block 10min in 3%H2O2 (in PBS) - Wash 3x in 1XPBST - 10min Avidin block - Wash 3x in 1XPBST - 10min. Biotin block - Wash 3x in 1XPBST - Surround tissue with DAKO pen - 7min. Superblock - Wash 3x in 1XPBST - 1° ab (PTEN) 1h at RT / on 4°C (1:200 in PBS 5% goat serum) - Wash 3x in 1XPBST - 10min. Biotinylated ab - Wash 3x in 1XPBS (NO TWEEN) - 10.min Streptav.-HRP - Wash 3x in 1XPBS (NO TWEEN) - AEC ( 20µl substrate +1000µl buffer) - stop in tab water - Wash in aqua dest. - Hemalaun - stop in tab water - mount with Aquatex |