Cytokine Bioassays Research Use Only

Transcription

1 Bioaay BetProtocol Page 1 of 1 Bioaay Introduction The biological activity of cytokine i commonly meaured by cellular proliferation of primary cell culture or in vitro adapted cell line that are dependent and/or reponive to a pecific growth factor. Other apect of biological activity of cytokine include induction of further cytokine ecretion, induction of killing, antiviral activity, degranulation, cytotoxicity, chemotaxi, and promotion of colony formation. In vitro aay to meaure all of thee activitie are available. Here, we dicu general protocol for etting up bioaay to meaure: Protocol A: -induced proliferation Protocol B: -induced killing Protocol C: Protection againt viral effect Protocol D: -induced cytokine production General Note Many of the available indicator cell line are reponive to more than one cytokine (e.g., CTLL cell repond to IL-, IL-4, IL-15) and everal cytokine hare ignaling receptor, o thi hould be taken into account during experimental deign. Neutralizing antibodie for one particular cytokine are ueful to demontrate origin of repone and attribute the activity to a particular cytokine. The dependence and reponivene of cell line hould be carefully acertained ince continuou culture of cell may alter their enitivity to growth factor. Bacterial endotoxin are effective inducer of ome cytokine; hence, their preence in the bioaay culture hould be prevented. In order to obtain tatitically ignificant value, ample hould be performed in triplicate well. Ueful webite Reearch Intitute ( Reearch Intitute i a webite maintained by Jutin Hoope at Utah State. Thi webite i to provide general information about the role cytokine play in dieae pathogenei. Reference ( Reference i a collection of information regarding cytokine and other mediator of immune repone. Revied Provided a a courtey by ebiocience, An Affymetrix Company Copyright ebiocience, Inc. Tel: or Fax: info@ebiocience.com

2 Bioaay BetProtocol Page of 1 Bioaay Protocol A: -induced proliferation Material cell line (ee Quick Guide Chart for a given cytokine) Culture (RPMI-1640 upplemented with 10% FBS) 96-well flat-bottom culture plate (Cotar Cat. No. 3595) MTT olution (Sigma Cat. No. M5655) 5mg/mL tock in PBS kept at room temperature (protect from light) MTT Lying Solution 0% SDS/50% DMF Intrument Pipette and pipettor Humidified incubator 96-well micro tet pectrophotometer Experiment Duration 4-48 hour incubation (ee Quick Guide Chart) 1 hour aay preparation Experimental Procedure 1. Add 100 µl of Culture to each well of the 96-well aay plate.. Dilute ample and tandard (ee Quick Guide Chart for tandard range) by -fold erial dilution in the aay plate from row to 1. Leave row 1 empty. 3. Wah indicator cell 3 time with RPMI-1640 and reupend in Culture at a denity of -3.5x10 5 cell/ml (ee Quick Guide Chart). 4. Add 100 µl of cell upenion to each well. 5. Incubate cell for 4-48 hour (ee Quick Guide Chart) at 37 C, 5% CO in a humidified incubator. 6. Add 10 µl/well of 5 mg/ml MTT olution to the plate and incubate for 4 hour. 7. Add 50 µl/well of MTT Lying Solution to the plate and incubate overnight. 8. Read plate at nm. 9. Graph tandard curve and analyze data. Protocol B: TNF-α-Induced Killing of L99 Line Material L99 moue fibroblat line Cat. No. CCL-1) Culture (RPMI upplemented with 10% FBS) (RPMI upplemented with % FBS) 96-well flat-bottom culture plate (Cotar Cat. No. 3595) Actinomycin D, 500 µg/ml tock aliquot kept at minu 80 C (protect from light) Revied Provided a a courtey by ebiocience, An Affymetrix Company Copyright ebiocience, Inc. Tel: or Fax: info@ebiocience.com

3 Bioaay BetProtocol Page 3 of 1 Bioaay MTT olution (Sigma Cat. No. M5655) 5 mg/ml tock in PBS kept at room temperature (protect from light) MTT Lying olution, 0% SDS/50% DMF Intrument Pipette and pipettor Humidified incubator 96-well micro tet pectrophotometer Experiment Duration 4-hour incubation (ee Quick Guide Chart) 1 hour aay preparation Experimental Procedure 1. Prepare L99 cell upenion at a denity of 3.5x10 5 /ml in. Add 100 µl/well to the 96-well aay plate and incubate overnight at 37 C, 5% CO in a humidified incubator.. Dilute ample and tandard (ee Quick Guide Chart for tandard range) by -fold erial dilution in the in 100 µl/well in another 96-well plate from row to 1. Leave row 1 empty. 3. Prepare a 4 µg/ml working olution of the Actinomycin D by diluting the 500 µg/ml tock 15 time in the. Keep Actinomycin D olution protected from light. Add 50 µl of thi working olution of Actinomycin D to each well. 4. Tranfer 50 µl of titrated ample and tandard to the correponding well on the aay plate. 5. Incubate plate for 4 hr at 37 C, 5% CO in a humidified incubator. 6. Add 10 µl/well of 5 mg/ml MTT olution to each well and incubate for 4 hour. 7. Add 50 µl of MTT Lying Solution to each well and incubate overnight. 8. Read plate at nm. Graph tandard curve and analyze data. Protocol C: IFN-γ Protection from Viral Infection of Line Material L99 moue fibroblat line Cat. No. CCL-1) or A549 human lung carcinoma Cat. No. CCL-185) Culture (RPMI upplemented with 10% FBS) (RPMI upplemented with % FBS) 96-well flat-bottom culture plate (Cotar Cat. No. 3595) EMC viru, 10 7 pfu/ml aliquot tock kept at minu 80 C MTT olution (Sigma Cat. No. M5655) 5 mg/ml tock in PBS kept at room temperature (protect from light) MTT Lying olution, 0% SDS/50% DMF Intrument Pipette and pipettor Humidified incubator 96-well micro tet pectrophotometer Revied Provided a a courtey by ebiocience, An Affymetrix Company Copyright ebiocience, Inc. Tel: or Fax: info@ebiocience.com

4 Bioaay BetProtocol Page 4 of 1 Bioaay Experiment Duration 40-hour incubation (ee Quick Guide Chart) 1 hour aay preparation Experimental Procedure 1. Prepare L99 or A549 cell upenion at a denity of 3.5x10 5 /ml in. Add 100 µl/well to the 96-well plate and incubate overnight at 37 C, 5% CO in a humidified incubator.. Dilute ample and tandard (ee Quick Guide Chart for tandard range) by -fold erial dilution in the aay plate from row to 1. Leave row 1 empty. Incubate the aay plate for 6 hour. 3. Apirate upernatant from the well carefully and add 100 µl/well of EMC viru olution at a denity of 1-4x10 4 pfu/ml (ee Quick Guide Chart for human IFN-γ: 1/50 dilution of tock of 10 7 pfu/ml, for moue IFN-γ, 1/1000 dilution of tock of 10 7 pfu/ml). 4. Incubate plate for 40 hour at 37 C, 5% CO in a humidified incubator. 5. Add 10 µl of 5 mg/ml MTT olution to each well and incubate for 4 hour. 6. Add 50 µl of MTT Lying Solution to each well and incubate overnight. 7. Read plate at nm. Graph tandard curve and analyze data. Protocol D: -Induced Production Material cell line (ee Quick Guide Chart for a given cytokine) Culture (RPMI-1640 upplemented with 10% FBS) 96-well flat-bottom culture plate (Cotar Cat. No. 3595) ELISA pair or Ready-SET-Go! Reagent Set Intrument Pipette and pipettor Humidified incubator 96-well micro tet pectrophotometer Experiment Duration 4-48-hour incubation (ee Quick Guide Chart) 1 hour aay preparation Experimental Procedure 1. Add 100 µl of Culture to each well of the 96-well aay plate.. Dilute ample and tandard (ee Quick Guide Chart for tandard range) by -fold erial dilution in the aay plate from row to 1. Leave row 1 empty. 3. Wah indicator cell 3 time with RPMI-1640 and reupend in Culture at a denity of -3.5x10 5 cell/ml (ee Quick Guide Chart). 4. Add 100 µl of cell upenion to each well. Revied Provided a a courtey by ebiocience, An Affymetrix Company Copyright ebiocience, Inc. Tel: or Fax: info@ebiocience.com

5 Bioaay BetProtocol Page 5 of 1 Bioaay 5. Incubate cell for 4-48 hour (ee Quick Guide Chart) at 37 C, 5% CO in a humidified incubator. 6. Harvet upernatant for determination of cytokine expreion by ELISA. 7. Follow ELISA protocol for target cytokine of interet. Quick Guide: Bioaay The following table ummarize the condition and reagent needed for Moue, Human and Rat cytokine bioaay. Moue Bioaay Quick Guide CCL5 (RANTE S) Chemeri n Incubatio n Time Specifi c Out/Comment CHOmCCR x10 3 Chemotaxi aay CHOmCMKLR Tranmigration x10 4 aay Flt3 IL-6 production, 1 Ligand M1 cell x10 4 ng/ml rmlif co-tim G-CSF M-NFS-60 rhm-csf (10 ng/ml) GM-CSF MC/9 CRL-8306) mil-3 (5 U/mL) Proliferation Proliferation HGF mimcd x x10 3 Proliferation HMGB1 RAW x x10 6 with 10 ug/ml of TNFa production; Polymycin B IFNα L99 CCL-1) EMCV cell IFNα4 L99 CCL-1) x EMCV cell IFN-γ L99 CCL-1) EMCV cell Revied Provided a a courtey by ebiocience, An Affymetrix Company Copyright ebiocience, Inc. Tel: or Fax: info@ebiocience.com

6 Bioaay BetProtocol Page 6 of 1 Bioaay IL-1α D10 TIB-4) IL-1α (10 pg/ml) + 10% rat ConA upe Proliferation IL-1β IL- IL-3 IL-4 IL-5 D10 TIB-4) CTLL- TIB-14) MC/9 CRL- 8306), NFS-60 CTLL- TIB-14) CRL-003) 3 mil-1α (10 pg/ml) hil- (100 U/mL) Incubatio n Time Specifi c Out/Comment Proliferation Proliferation mgm-csf Proliferation hil- (100 U/mL) (100 U/mL) Proliferation Proliferation IL-6 B9 hil-6 (50 pg/ml) Proliferation; Starve cell for 48 hr prior IL-7 hpbmc x Proliferation; PHA (5 ug/ml) a cotimulator IL-9 TS-1 IL-10 IL-1 p70 IL-13 IL-15 MC/9 CRL-8306) plenocyte CRL-003) CTLL- TIB-14) rmil-9 (100 ng/ml) Proliferation mgm-csf x Proliferation; mil-4 (5 pg/ml) a cotimulator x mifn-γ production (100 U/mL) rmil- (100 ng/ml) x ,800 Proliferation x10 4 Proliferation Revied Provided a a courtey by ebiocience, An Affymetrix Company Copyright ebiocience, Inc. Tel: or Fax: info@ebiocience.com

7 Bioaay BetProtocol Page 7 of 1 Bioaay IL- 15R/IL- 15 CTLL- TIB-14) rmil- (100 ng/ml) x10 3 Proliferation IL-17A NIH/3T3 CRL-1658) x IL-6 production IL-17A/F IL-17F NIH/3T3 CRL-1658) NIH/3T3 CRL-1658) x10 4 IL-6 production Incubatio n Time Specifi c Out/Comment x IL-6 production.5x10 IL-19 RAW x10 6 IL-6 production IL-1 B Proliferation IL- Colo IL-10 production IL-3 plenocyte mil-17 production; PMA (5 ng/ml) a cotimulator IL-7 plenocyte x Inhibition of IL- ecretion; a cotimulator IL-8 HEPG EMCV cell x10 IL-33 D10.G Proliferation LIF M IL-6 production M-CSF M-NFS-60 SCF MC/9 CRL-8306) rhm-csf (10 ng/ml) mgm-csf (100 ng/ml) x10 3 Proliferation 7 6x x10 4 Proliferation Sonic Hedgeho g (SHH) C3H/10T1/ 3 10 (heat inactivated FBS) 7 day.5x Alkaline phophatae induction TNF-α L99 CCL-1) x L99 cytotoxicity aay; Actinomycin D ( ug/ml) TSLP Nag8/7 rmil Proliferation Revied Provided a a courtey by ebiocience, An Affymetrix Company Copyright ebiocience, Inc. Tel: or Fax: info@ebiocience.com

8 Bioaay BetProtocol Page 8 of 1 Bioaay VEGF Human umbilical vein endothelial cell (10 ng/ml) rhvegf (100 U/mL) HGF: hepatocyte growth factor ED 50: 50% effective doe of cytokine (1 U/mL oberved activity) x10 3 Proliferation Table 1: Moue Bioaay Quick Guide Human Bioaay Quick Guide Incubation Time Specific ActivinA MPC BMP- CCL5 (RANTES) C3H/10T1/ 3 10 (heat inactivated erum) 1 week x10 6 CHOmCCR Out/Comment Inhibition of proliferation Alkaline phophatae induction Chemotaxi FGF8 Balb/c-3T FGFa Balb/c-3T Proliferation; 10 ug/ml Heparin co-tim FGFb Balb/c-3T Proliferation Flt3-L M G-CSF M-NFS-60 GM-CSF HGF IFN-α1 IFN-α CRL-003) 4MBr-5 CCL-08) A549 CCL-185) A549 CCL-185) rhm-csf (10 ng/ml) Proliferation; x10 4 ug/ml Heparin co-tim IL-6 production; x10 4 ng/ml rmlif cotim Proliferation (100 U/mL) Proliferation rmegf (30 ng/ml) x x10 4 Proliferation x x A549/EMCV cell A549/EMCV cell Revied Provided a a courtey by ebiocience, An Affymetrix Company Copyright ebiocience, Inc. Tel: or Fax: info@ebiocience.com

9 Bioaay BetProtocol Page 9 of 1 Bioaay IFN-γ IL-1α IL-1β IL- A549 CCL-185) D10 TIB-4) D10 TIB-4) CTLL- TIB-14) A549/EMCV cell mil-1α (10 pg/ml) x Proliferation IL-1α (10 pg/ml) + 10% rat ConA upe Proliferation hil- (100 U/mL) Proliferation Incubation Time Specific Out/Comment IL-3 CRL-003) ( ng/ml) x Proliferation IL-4 CRL-003) (100 U/mL) Proliferation IL-5 CRL-003) (100 U/mL) Proliferation IL-6 CRL-003) (100 U/mL) Proliferation IL-7 IL-8 Human PHAtreated PBMC (3day) Human Neutrophil x ,000 Proliferation IL-10 MC/9 CRL-8306) mcm-csf Proliferation IL-1 p70 hpbmc (PHA blat) x Proliferation IL-13 CRL-003) (100 U/mL) x10 3 Proliferation Revied Provided a a courtey by ebiocience, An Affymetrix Company Copyright ebiocience, Inc. Tel: or Fax: info@ebiocience.com

10 Bioaay BetProtocol Page 10 of 1 Bioaay IL-15 CTLL- TIB-14) 3 IL- (100 U/mL) Proliferation IL-17A NHDF x ,000 IL-6 production IL-17A/F NIH/3T x10 4 IL-6 production IL-17F NIH/3T x10 6 IL-6 production Incubation Time Specific Out/Comment IL-19 hpbmc x10 5 IL-6 production IL-1 B9 hil-6 (50 pg/ml) Proliferation IL- Colo IL-10 production IL-3 Moue plenocyte x x10 3 production; cotim w/ TPA (5 mil-17 ng/ml) IL-6 COLO x10 4 IL-10 production IL-7 Moue plenocyte x Inhibition of IL- 1.5x10 4 ecretion 3 EMCV cell IL-8B HepG x10 IL-9 HepG EMCV cell IL-31 U STAT3 activation IL-33 D10.G Proliferation M-CSF M-NFS-60 rhm-csf (10 ng/ml) x10 3 Proliferation SCF CRL-003) (100 U/mL) x ,000 Proliferation Revied Provided a a courtey by ebiocience, An Affymetrix Company Copyright ebiocience, Inc. Tel: or Fax: info@ebiocience.com

11 Bioaay BetProtocol Page 11 of 1 Bioaay TGF-β1 HT- (CRL- 1841) rmil- (100 U/mL) Inhibition of IL-4 induced proliferation TGF-β CTLL- IL- (100 ng/ml) Inhibition of IL-4 induced proliferation TGF-β3 CTLL- IL- (100 ng/ml) Inhibition of IL-4 induced proliferation TNF-α L99 CCL-1) x L99 cytotoxicity aay; Actinomycin D ( ug/ml) Incubation Time Specific Out/Comment TSLP hpbmc </=10 4 htarc induction VEGF HUVEC rhvegf (100 U/mL) ,000 Proliferation HGF: hepatocyte growth factor ED 50: 50% effective doe of cytokine (1 U/mL oberved activity) Table : Human Bioaay Quick Guide Rat Bioaay Quick Guide Incubation Time Specific IFN-γ L IL-1β IL- IL-4 D10 TIB-4) CTLL- TIB-14) plenocyte Out/Comment EMCV cell protection aay mil-1α (10 pg/ml) Proliferation hil- (100 U/mL) x Proliferation x10 3 Proliferation IL-17A NIH/3T x x10 4 Moue IL-6 production Revied Provided a a courtey by ebiocience, An Affymetrix Company Copyright ebiocience, Inc. Tel: or Fax: info@ebiocience.com

12 Bioaay BetProtocol Page 1 of 1 Bioaay IL-17AF NIH/3T x10 4 Moue IL-6 production IL-17F NIH/3T x x10 6 Moue IL-6 production TNF-α L99 CCL-1) x L99 cytotoxicity aay; Actinomycin D ( ug/ml) ED 50: 50% effective doe of cytokine (1 U/mL oberved activity) Table 3: Rat Bioaay Quick Guide Revied Provided a a courtey by ebiocience, An Affymetrix Company Copyright ebiocience, Inc. Tel: or Fax: info@ebiocience.com