Sample Cytotoxicity Assay Protocol

Transcription

1 Sample Cyttxicity Assay Prtcl Plating (Example) Day 1: Plate cells at 2500 cells/well fr a typical 3- plate cyttxicity assay: Cells shuld be lifted frm the flask (if need be) and cunted. Only the center 60 wells have cells added t them. Add 100 µl f culture media t the uter ring f wells t avid evapratin f the sample wells during the curse f the assay. Fr the present example, the calculatin fr the number f cells needed is as fllws: Number f wells Cells/well Ttal cells needed 200 x ,000 µl/well Ttal µl media 200 x 90 18,000 The cells will need t be resuspended very thrughly, and create a single cell suspensin as best as pssible via a cmbinatin f pipetting up and dwn plus vrtexing. Add 100 micrliters f media ONLY t each f the uter ring f cells. Using a multi- channel pipettr quickens the prcess. Pur the cell suspensin int a reservir that is easy t pipet frm. Add 90 micrliters f the cell suspensin t each f the center 60 wells n each plate. Label each plate in sme fashin with the cell type, # f cells/well, and date. Place the plates int the prper culture envirnment vernight. SAPORIN Preparing Saprin cntrl dilutins (example) Day 2: Cntrls and samples shuld be added t the plates in the mrning (~16 hurs after the cells are plated). Saprin dilutins will be prepared at 8 cncentratins, diluted frm 1 µm t 1 pm, in 1:10 dilutin increments.

2 Cyttxicity Assay 2 These are final cncentratins in the well, but will be added t the well in a 10 µl vlume, therefre the beginning cncentratin will be 10- fld higher, 10 µm. View calculatins belw, fr Saprin at a cncentratin f 1.0 mg/ml, 45 micrliters f material will be used. In a micrcentrifuge rack, line up 8 tubes, labeling them 1-8. Add 135 µl f cell culture media t tubes 2-7. T the first tube, add 105 µl f culture media (150 µl 45 µl). Cnc. (µm) vlume (µl) Saprin Cnc. Saprin Mlec E+04 Vlume f Saprin X E- 05 M X µm 45 µl Add 45 µl f Saprin t tube #1, bringing the final vlume t 150 µl. Perfrm serial dilutins by pipetting 15 µl frm tube 1 int tube 2. Pipet up and dwn several times t mix. Vrtex the tube briefly. Repeat by pipetting 15 µl frm tube 2 int tube 3. Cntinue t repeat the steps thrugh tube 8. Whle- ZAP, Fab- ZAP, & FabFc- ZAP Preparing ZAP spiked media (example; Whle, Fab, & FabFc ZAP prducts) Day 2: These three ZAP prduct types are used at a cnstant cncentratin within the assay. Ding s ensures that the effects witnessed in the assay are attributable t the targeting agent alne. The recmmended effective cncentratin is 4.5 nanmlar in the wells. In rder t best titrate the targeting agent, it is suggested that the media used fr the titratin be spiked with the ZAP prduct at 45 nm such that a 1:10 dilutin during additin t the wells results in a prper final cncentratin. Cnc. (nm) vlume (µl) ZAP Cnc. ZAP Mlec. Tube Label Data Sheet Vlume f ZAP X

3 3 Cyttxicity Assay M X nm µl Add the calculated vlume f ZAP prduct t 1.5 ml f culture media and vrtex. Label tube as ZAP media, r smething similar, s as nt t mix it up with the regular culture media. Preparing Targeting Agent (test sample) dilutins (example) Day 2: Samples shuld be added t the plates in the mrning (~16 hurs after the cells are plated). Targeting Agent dilutins will be prepared at 8 cncentratins, diluting frm 10 nm t 1 fm, in 1:10 dilutin increments. These are final cncentratins in the well, but will be added t the well in a 10 µl vlume, therefre the beginning cncentratin will be 10- fld higher, 100 nm. View calculatins belw fr Yur Targeting Agent. Input yur knwn cncentratin and mlecular weight fr the agent. In a micrcentrifuge rack, line up 8 tubes, labeling them 1-8. Add 135 µl f ZAP- spiked culture media t tubes 2-7. T the first tube, add 150 µl f ZAP- spiked culture media. Cnc. (µm) vlume (µl) Sample Cnc. Sample Mlec. mg/ml Da Vlume f ZAP X 15 M X 15 µm µl Remve the calculated vlume f ZAP spiked media frm tube 1 and discard. Add the calculated vlume f Targeting Agent t tube 1, bringing the final vlume t 150 µl. Immediately perfrm serial dilutins by pipetting 15 µl frm tube 1 int tube 2. Pipet up and dwn several times t mix. Vrtex the tube briefly. Repeat by pipetting 15 µl frm tube 2 int tube 3. Cntinue t repeat the steps thrugh tube 8.

4 Cyttxicity Assay 4 Allw tubes t incubate at rm temperature fr 15 minutes. CONTROL- SAP Preparing Cntrl samples (example) Day 2: Samples shuld be added t the plates in the mrning (~16 hurs after the cells are plated). Cntrl- SAP dilutins will be prepared at 8 cncentratins, diluting frm 10 nm t 1 fm, in 1:10 dilutin increments. These are final cncentratins in the well, but will be added t the well in a 10 µl vlume, therefre the beginning cncentratin will be 10- fld higher, 100 nm. View calculatins belw, fr Cntrl sample using the cncentratin n the tube label and mlecular weight given n the data sheet. In a micrcentrifuge rack, line up 8 tubes, labeling them 1-8. Add 135 µl f regular culture media t tubes 2-7. T the first tube, add 150 µl f regular culture media. Cnc. (µm) vlume (µl) Cntrl Cnc. Cntrl Mlec. Tube Label Data Sheet Vlume f ZAP X 15 M X 15 µm µl Remve the calculated vlume f culture media frm tube #1 and discard. Add the calculated vlume f Cntrl sample t tube #1, bringing the final vlume t 150 µl. Perfrm serial dilutins by pipetting 15 µl frm tube 1 int tube 2. Pipet up and dwn several times t mix. Vrtex the tube briefly. Repeat by pipetting 15 µl frm tube 2 int tube 3. Cntinue t repeat the steps thrugh tube 8.

5 5 Cyttxicity Assay TREATMENT Adding Samples t the Plates (example) Day 2: This kit is designed t be used with three 96- well plates. One plate fr Saprin, ne plate fr Cntrl- SAP, and ne plate fr the Targeting Agent sample in cnjunctin with the included ZAP prduct. It is recmmended that the lid f the plate be labeled with the reagents t be added t the plate. All materials are added t the plates in 10 µ l vlumes. Samples are added t the plate in 6 replicates, ne cncentratin per well- clumn. Traditinally plate- clumn 2 and 11 have culture media ONLY added, 10 µ l per well. If using Whle, Fab, r FabFc- ZAP spiked media, add 10 ul f ZAP media alne t plate- clumn 11 as an internal cntrl. Incubate all plates under nrmal culture cnditins fr 72 hurs. RESULTS Develping the Assay (example) Day 5: Warm 5.5 milliliters f PBS in a 15 ml cnical tube t 37ºC. Thaw XTT vial t rm temp and vrtex thrughly. Add entire cntents f XTT tube t pre- warmed PBS and vrtex again. Add 92 micrliters f PMS t the XTT/PBS tube and vrtex thrughly. Add 50 µl f the XTT/PMS slutin t each f the interir 60 wells and the A1 blank well r each plate. Incubate plates at 37ºC. Plates shuld be read apprximately every 30 minutes t determine assay cmpletin. (Typical ttal incubatin time is 2 hurs, but this will vary by cell type) Advanced Targeting Systems

6 Cyttxicity Assay 6 T read plates, shake gently fr 10 secnds in plate reader prir t reading. Read the plates at 450 nm. Optical density readings fr the cntrl wells shuld be >0.3 fr best results. Sample Data Presentatin: Cyttxicity data is typically analyzed by cmparing well readings f the treated wells t thse f the cntrl wells, expressed as a percentage. The number f viable cells remaining n the day f develpment is measured via cell metablism f a clrimetric mlecule within the develping reagents. The darkness f clr in the untreated wells is cnsidered t be 100% f cntrl. Materials & Safety Gd labratry technique must be emplyed fr the safe handling f this prduct. This requires bservatin f the fllwing practices: Wear apprpriate labratry attire, including lab cat, glves and safety glasses. D nt pipet by muth, inhale, ingest r allw prduct t cme int cntact with pen wunds. Wash thrughly any part f the bdy which cmes int cntact with the prduct. Avid accidental autinjectin by exercising extreme care when handling in cnjunctin with any injectin device. This prduct is intended fr research use by qualified persnnel nly. It is nt intended fr use in humans r as a diagnstic agent. Advanced Targeting Systems is nt liable fr any damages resulting frm the misuse r handling f this prduct. See data sheets enclsed with kit fr individual cmpnent safety and handling infrmatin. References Khls MD, Lappi DA (2000) MabZAP: A tl fr evaluating antibdy efficacy fr use in an immuntxin. BiTechniques 28(1): Advanced Targeting Systems

7 Cyttxicity Assay 7 Ordering Yur Ab Available ZAP Prducts chicken IgY gat IgG Chick- ZAP (IT- 62) Gat- ZAP (IT- 36) guinea pig IgG gpig- ZAP (IT- 64) human IgG Hum- ZAP (IT- 22) Fab- ZAP human (IT- 51) FabFc- ZAP human (IT- 65) human IgM Hug- M- ZAP (IT- 43) muse IgG Mab- ZAP (IT- 04) Fab- ZAP muse (IT- 48) muse IgM Anti- M- ZAP (IT- 30) rabbit IgG Rab- ZAP (IT- 05) Fab- ZAP rabbit (IT- 57) rat IgG Rat- ZAP (IT- 26) Fab- ZAP rat (IT- 55) All available individually (100 µg r 250 µg) r as a kit made with bivalent antibdies that recgnize the whle IgG made with mnvalent (Fab) antibdies that recgnize the whle IgG, withut bivalent capping made with mnvalent (Fab) antibdies that recgnize Fc regin nly