Descripción del Protocolo
Disuelva 10 mg de zymosan de Saccharomyces cerevisiae (Sigma Z4250) en 50 ml de PBS estéril y luego diluya 10 veces para obtener una solución de trabajo de 20 mg/ml.
Autoclave a 121ºC durante 20 min.
Mantenga el zimosan esterilizado en autoclave en hielo.
Inyectar 0,5 ml de zymosan i.p. (10 mg/cavidad peritoneal del ratón; mezcle zymosan inmediatamente antes de la inyección para garantizar una suspensión uniforme) con una aguja 25G 5/8.
Esperar 4 h.
Sacrificar ratones por exposición a dióxido de carbono.
Realice una pequeña incisión (~0,5 cm) a lo largo de la línea media del abdomen que permita retirar la piel para exponerla el abdomen, que luego se rocía con etanol al 70%.
Inyecte 3 ml de solución de lavado de PBS/EDTA 2 mM helada con una jeringa y una aguja de 25G 5/8.
Masajee suavemente el abdomen del ratón con la longitud de una aguja de 11⁄2 de 18G para asegurarse de que las células sueltas adheridos a la pared peritoneal u otros órganos se desprenderán y quedarán suspendidos en el líquido de lavado.
Use una aguja 18G 11⁄2 para extraer suave y lentamente el líquido de lavado de la cavidad peritoneal. Transfiera el líquido a un tubo de centrífuga de 15 ml y manténgalo en hielo. Es posible que no obtenga los 3 ml debido a los volúmenes muertos en el peritoneo.
Transferir 200\(\mu\)l células a un tubo cónico de 15 mL, centrifugar y desechar el sobrenadante.
Teñir las células con los anticuerpos apropiados (CD45-PerCP, F4/80-PE/Cy7, Ly6G-APC) durante 15 min en hielo.
Lavar las células con tampón de tinción, seguido de resuspensión en 300\(\mu\)l de buffer de tinción. Agregar 30\(\mu\)l de perlas de conteo (30,000 partículas)
Ejecutar en el flujo.
Conocemos el número total de cuentas, por lo que los números absolutos de celdas se pueden calcular en relación con las cuentas. Debe multiplicar 15 veces, ya que de 3 mL se utilizaron 200 \(\mu\)l de exudado. (Número absoluto de celdas) = (recuento de celdas) / (recuento de perlas) X 30 000 X 15
Diagrama de Flujo
Anexos
Soluciones
Reactivos
- Zymosan de Saccharomyces cerevisiae Sigma Z4250
Documentos de ?@sec-ref
old
Zymosan-‐induced
peritonitis
protocol Feb 15, 2012 Wooki Kim 1. Dissolve 10 mg zymosan from Saccharomyces cerevisiae (Sigma Z4250) in 50 ml sterile PBS then dilute 10x to obtain a 20 mg/ml working solution. 2. 3. 4. Autoclave at 121oC for 20 min. Keep autoclaved zymosan on ice. Inject 0.5 ml zymosan i.p. (10 mg/mouse peritoneal cavity; mix zymosan immediately before injection to ensure an even suspension) with a 25G 5/8 needle. 5. Wait for 4 hr. 6. 7. Sacrifice mice by exposure to a carbon dioxide. Make a small (~0.5 cm) incision along the midline of the abdomen allowing the skin to be pulled back to expose the abdomen, which is then sprayed with 70% ethanol. 8. 9. Inject 3 ml ice-cold PBS/2 mM EDTA washing solution with a syringe and 25G 5/8 needle. Gently massage the mouse abdomen with the length of a 18G 1½ needle to ensure that cells that are loosely adherent to the peritoneal wall or other organs will detach and become suspended in the lavage fluid. 10. Use a 18G 1½ needle to gently and slowly extract the lavage fluid from the peritoneal cavity. Transfer the fluid to a 15-ml centrifuge tube and keep on ice. You may not harvest all 3 mL due to the dead volumes in peritoneum. 11. Transfer 200 µL cells to a 15 mL conical tube, centrifuge, then discard the supernatant. 12. Stain cells with appropriate antibodies (CD45-PerCP, F4/80-PE/Cy7, Ly6G-APC) for 15 min on the ice. 13. Wash cells with staining buffer, followed by resuspension into 300 uL staining buffer. Add 30 uL of counting beads (30,000 particles) 14. Run on flow. 15. We know the total number of beads, so that absolute cell numbers can be calculated relative to the beads. You should multiply 15 times, since 200 µL of exudate was used out of 3 mL. (Absolute cell number) = (cell count) / (count beads) X 30,000 X 15
competititon
PMN competition
protocol Feb 12, 2012 Wooki Kim Zymosan preparation 1. 2. 3. Dissolve 10 mg zymosan from Saccharomyces cerevisiae (Sigma Z4250) in 50 ml sterile PBS then dilute 10x to obtain a 20 mg/ml working solution. Autoclave at 121oC for 20 min. Keep autoclaved zymosan on ice. Bone marrow harvest 1. Immediately before surgery, sacrifice mice with CO2. 2. Spray all external areas of the mouse with 70% ethanol. 3. Make an incision on the abdomen. 4. Gently pull the skin downward below the heels to expose the muscles, etc. 5. Using a sharp scissor, dissect femurs from surrounding muscles and tendons. 6. Place the femurs into a petri dish with 20 mL DPBS on ice. 7. Using a sharp scissor and paper towels, remove excess tissue. 8. Cut both ends of femurs. 9. Fill a 10 mL syringe with DPBS, then cap with a 25G 5/8 gauge needle. 10. Drill the needle into the open end of the femur. 11. Flush DPBS through the femur. 12. Using the syringe without a needle, pipet up/down bone marrow clots into single cells suspension. 13. Place a 100 µm mesh on a 50 mL conical tube, and pass the cell suspension through. 14. Spin down cells, aspirate supernatants, and keep bone marrow cells on ice. Count cells/mix 1. 2. 3. 4. 5. 6. Mix 180 µL ACK lysis buffer + 10 µL Trypan blue + 10 µL cell suspension. Wait 5 min for red cell lysis. Determine cell numbers by using hemocytometer. Adjust cell concentration at 1 x 107 cells/mL. Mix JAM-A KO cells with CD45.1 cells or WT cells with CD45.1 cells at 1:1 ratio. Transfer cell mixture into a 1 mL syringe with a needle. i.v. injection to host mice 1. 2. Heat CD45.1/CD45.2 heterozygous host mice (at the age of 4-6 wk, preferably) under an incandescent light. Adoptively transfer 200 µL cell mixture per mouse via a tail vein. Peritonitis by zymosan 1. Inject 0.5 ml autoclave zymosan i.p. (10 mg/mouse peritoneal cavity; mix zymosan immediately before injection to ensure an even suspension) with a 25G 5/8 needle. 2. Wait for 4 hr. Peritoneal lavage 1. 2. 3. 4. Sacrifice mice by exposure to a carbon dioxide. Make a small (~0.5 cm) incision along the midline of the abdomen allowing the skin to be pulled back to expose the abdomen, which is then sprayed with 70% ethanol. Inject 3 ml ice-cold PBS/2 mM EDTA washing solution with a syringe and 25G 5/8 needle. Gently massage the mouse abdomen with the length of an 18G 1½ needle to ensure that cells that are loosely adherent to the peritoneal wall or other organs will detach and become suspended in the lavage fluid. Use an 18G 1½ needle to gently and slowly extract the lavage fluid from the peritoneal cavity. Transfer the fluid to a 15-ml centrifuge tube and keep on ice. You may not harvest all 3 mL due to the dead volumes in peritoneum. Surface staining and Flowcytometry (LSR II) 1. 2. 3. 4. 5. Transfer 200 µL cells to a 15 mL conical tube, centrifuge, then discard the supernatant. Stain cells with appropriate antibodies (Ly6G-APC, CD45.1-FITC, CD45.2-PE) for 15 min on the ice. Wash cells with staining buffer, followed by resuspension into 300 uL staining buffer. Collect until the total events reach 1,000,000. Gating strategy for data analysis: Ly6G vs SSC-H CD45.1 vs CD45.2 5.