T-solujen aktivaatio Concanavalin A: n kautta

kirjoittaja

Johdanto

T-solut vaativat ulkoisia signaaleja erilaistumiseen ja laajentumiseen lepotilasta. Konkanavaliini A (con A) on antigeenistä riippumaton mitogeeni ja sitä voidaan käyttää vaihtoehtoisena T-solun ärsykkeenä. Tätä lektiiniä käytetään usein antigeeniä esittelevien solujen korvikkeena T-solujen stimulaatiokokeissa. Konkaanavaliini A sitoutuu peruuttamattomasti solun pinnalla oleviin glykoproteiineihin ja sitouttaa T-solut lisääntymään. Tämä on nopea tapa stimuloida transkriptiotekijöitä ja sytokiinituotantoa.


Materiaalit

  1. Steriilit T25- tai T75-pullot, joissa on tuuletetut korkit (esim. Nunc EasYFlask -soluviljelypullot, T25, suodatin, luettelonro 156367)
  2. RPMI 1640 medium (esim. BenchStable RPMI 1640 Medium, luettelonro A4192301)
  3. Naudan sikiön seerumi (esim. Gibco Fetal Bovine Serum, luettelonro 26140087 tai Fetal Bovine Serum One Shot -muoto, luettelonro A3160401)
  4. L-glutamiini (esim. 200 mM L-glutamiini, luettelonro 25030149)
  5. Valinnainen: penisilliini-streptomysiini (esim. Gibco Penicillin-Streptomycin, luettelonro 15140148)
  6. Concanavalin A (esim. EBioscience Concanavalin A -liuos, 500X, luettelonro 00-4978-93)
  7. Solunkaavin (esim. Nunc Cell Scrapers, luettelonro 179693)


Menettely

  • Käyttämällä aseptisia tekniikoita steriileissä olosuhteissa, eristää PBMC kokoverestä (lisäprotokolla A) tai valmistetusta imukudoksesta (lisäprotokolla B).
  • Valmista täydellinen RPMI 1640 -elatusaine lisäämällä RPMI 1640 -elatusaine naudan sikiöseerumilla lopulliseen konsentraatioon 10% ja 2 mM L-glutamiinia (jos käytetään väliainetta, jota ei tällä hetkellä ole täydennetty GlutaMAX: lla). Tuo väliaine 37 ° C: seen. Valinnainen:  Täytä elatusaine 1% penisilliini-streptomysiinillä (5000 yksikköä / ml).
  • Solujen suspendoimiseksi konsentraatioon 3 x 10 6  solua / ml täydelliseen RPMI 1640-väliaine. Valmista tarvittava tilavuus käyttämäsi pullon koon perusteella (T25 tai T75).
  • Valmista 2 viljelypulloa: yksi merkitty “con A stimulated” (aktivoitu) ja toinen “stimuloimaton” (ei aktivoitu).
  • Jaa soluliuos (valmistettu vaiheessa 3) tasaisesti valmistettuihin pulloihin.
  • Lisää konkanavaliini A -liuosta konsentraatiossa 1X (2 ui / ml) conA-stimuloituun (aktivoituun) pulloon.
  • Peitä molemmat pullot suodatetuilla tuuletetuilla korkkeilla ja inkuboi pulloja 1-3 päivän ajan 5-prosenttisessa CO 2  -inkubaattorissa 37 ° C: ssa.
  • Sadonkorjuu soluja kaapimella.


Lisäprotokolla A: PBMC: n eristäminen kokoverestä

Materiaalit

  1. Fosfaattipuskuroitu suolaliuos (esim. Gibco PBS (10X), pH 7,4, luettelonro 70011044)
  2. 15 ml tai 50 ml kartiomainen putki (esim. Nunc 15 ml kartiomaiset steriilit sentrifugiputket, luettelonro 339650)
  3. Ficoll-   tiheys erotusvälineen
  4. Virtaussytometrinen värjäyspuskuri (esim. EBioscience Flow Cytometry -värjäyspuskuri, luettelonro 00-4222-26)

Menettely

  • Laimenna verinäyte vähintään 1: 1 PBS: llä kartiomaisessa putkessa.
  • Laita laimennettu näyte Ficoll-Paque  -alustan tilavuuteen,  joka on yhtä suuri kuin alkuperäinen näytetilavuus.
  • Sentrifugoidaan nopeudella 400 x g 20 minuuttia huoneenlämmössä jarrutuksen ollessa POIS.
    • Kerää PBS: n ja Ficoll-Paque  -alustakerrosten rajapinnalla oleva PBMC  tuoreeseen putkeen.
  • Täytä putki PBS: llä solujen pesemiseksi.
  • Sentrifugoi soluja 300–400 xg: llä 4–5 minuuttia 2–8 ° C: ssa. Hävitä supernatantti.
  • Suspendoi solupelletti uudelleen sopivaan tilavuuteen virtaussytometristä värjäyspuskuria tai valittua puskuria ja suorita solumäärä- ja elinkelpoisuusanalyysi.
  • Sentrifugoi solut kuten vaiheessa 6, ja suspendoi uudelleen sopivaan tilavuuteen täydellistä RPMI 1640: tä niin, että lopullinen solupitoisuus on 3 x 106  solua / ml.

Lisäprotokolla B: immuunisolujen eristäminen imukudoksesta

Materiaalit

  1. 60 x 15 mm: n soluviljelymalja (esim. Nunc-soluviljely Petri-astiat, luettelonro 150340)
  2. Muovinen 3 ml: n ruisku tai kaksi himmeä lasimikroskoopin lasia
  3. Solusihti (nailonverkko)
  4. 15 ml tai 50 ml kartiomainen putki (esim. Nunc 15 ml kartiomaiset steriilit sentrifugiputket, luettelonro 339650)
  5. Virtaussytometrinen värjäyspuskuri (esim. EBioscience Flow Cytometry -värjäyspuskuri, luettelonro 00-4222-26).

 

Rac1 Activation Assay
STA-401-1 20 assays
EUR 908.4
Description: Our Rac Activation Assays use visible agarose beads to selectively precipitate the active form of Rac1 or Rac2. The precipitated small GTPase is then detected by Western blot using a Rac1- or Rac2-specific antibody included in the kit.

Rac2 Activation Assay
STA-401-2 20 assays
EUR 908.4
Description: Our Rac Activation Assays use visible agarose beads to selectively precipitate the active form of Rac1 or Rac2. The precipitated small GTPase is then detected by Western blot using a Rac1- or Rac2-specific antibody included in the kit.

RhoA Activation Assay
STA-403-A 20 assays
EUR 908.4
Description: Our Rho Activation Assays use visible agarose beads to selectively precipitate the active form of RhoA, RhoB or RhoC. The precipitated small GTPase is then detected by Western blot using a RhoA-, RhoB- or RhoC-specific antibody included in the kit.

RhoB Activation Assay
STA-403-B 20 assays
EUR 908.4
Description: Our Rho Activation Assays use visible agarose beads to selectively precipitate the active form of RhoA, RhoB or RhoC. The precipitated small GTPase is then detected by Western blot using a RhoA-, RhoB- or RhoC-specific antibody included in the kit.

RhoC Activation Assay
STA-403-C 20 assays
EUR 908.4
Description: Our Rho Activation Assays use visible agarose beads to selectively precipitate the active form of RhoA, RhoB or RhoC. The precipitated small GTPase is then detected by Western blot using a RhoA-, RhoB- or RhoC-specific antibody included in the kit.

Arf1 Activation Assay
STA-407-1 20 assays
EUR 908.4
Description: Our Arf Activation Assays use visible agarose beads to selectively precipitate the active form of Arf1 or Arf 6. The precipitated small GTPase is then detected by Western blot using an Arf1- or Arf6-specific antibody included in the kit.

Arf6 Activation Assay
STA-407-6 20 assays
EUR 908.4
Description: Our Arf Activation Assays use visible agarose beads to selectively precipitate the active form of Arf1 or Arf 6. The precipitated small GTPase is then detected by Western blot using an Arf1- or Arf6-specific antibody included in the kit.

Cdc42 Activation Assay
STA-402 20 assays
EUR 908.4
Description: Our Cdc42 Activation Assays use visible agarose beads to selectively precipitate the active form of Cdc42 protein. The precipitated small GTPase is then detected by Western blot using a Cdc42-specific antibody included in the kit.

H-Ras Activation Assay
STA-400-H 20 assays
EUR 908.4
Description: Our Ras Activation Assays use visible agarose beads to selectively precipitate the active form of specific Ras protein of interest. The precipitated small GTPase is then detected by Western blot using a target-specific antibody included in the kit. Assays are available to detect specific isoforms H-Ras, K-Ras, and N-Ras, as well as a Pan-Ras assay that detects all three isoforms.

K-Ras Activation Assay
STA-400-K 20 assays
EUR 908.4
Description: Our Ras Activation Assays use visible agarose beads to selectively precipitate the active form of specific Ras protein of interest. The precipitated small GTPase is then detected by Western blot using a target-specific antibody included in the kit. Assays are available to detect specific isoforms H-Ras, K-Ras, and N-Ras, as well as a Pan-Ras assay that detects all three isoforms.

N-Ras Activation Assay
STA-400-N 20 assays
EUR 908.4
Description: Our Ras Activation Assays use visible agarose beads to selectively precipitate the active form of specific Ras protein of interest. The precipitated small GTPase is then detected by Western blot using a target-specific antibody included in the kit. Assays are available to detect specific isoforms H-Ras, K-Ras, and N-Ras, as well as a Pan-Ras assay that detects all three isoforms.

Rap1 Activation Assay Kit
STA-406-1 20 assays
EUR 908.4
Description: Our Rap Activation Assays use visible agarose beads to selectively precipitate the active form of Rap1 or Rap2. The precipitated small GTPase is then detected by Western blot using a Rap1- or Rap2-specific antibody included in the kit.

Rap2 Activation Assay Kit
STA-406-2 20 assays
EUR 908.4
Description: Our Rap Activation Assays use visible agarose beads to selectively precipitate the active form of Rap1 or Rap2. The precipitated small GTPase is then detected by Western blot using a Rap1- or Rap2-specific antibody included in the kit.

Pan-Ras Activation Assay
STA-400 20 assays
EUR 908.4
Description: Our Ras Activation Assays use visible agarose beads to selectively precipitate the active form of specific Ras protein of interest. The precipitated small GTPase is then detected by Western blot using a target-specific antibody included in the kit. Assays are available to detect specific isoforms H-Ras, K-Ras, and N-Ras, as well as a Pan-Ras assay that detects all three isoforms.

Ral Activation Assay Kit, Trial Size
STA-408-T 5 assays
EUR 483.6
Description: Our Ral Activation Assay uses visible agarose beads to selectively precipitate the active form of Ral protein. The precipitated small GTPase is then detected by Western blot using a Ral-specific antibody included in the kit.

Ran Activation Assay Kit, Trial Size
STA-409-T 5 assays
EUR 483.6
Description: Our Ran Activation Assay uses visible agarose beads to selectively precipitate the active form of Ran protein. The precipitated small GTPase is then detected by Western blot using a Ran-specific antibody included in the kit.

Rac1 Activation Assay Kit, Trial Size
STA-401-1-T 5 assays
EUR 483.6
Description: Our Rac Activation Assays use visible agarose beads to selectively precipitate the active form of Rac1 or Rac2. The precipitated small GTPase is then detected by Western blot using a Rac1- or Rac2-specific antibody included in the kit.

Rac2 Activation Assay Kit, Trial Size
STA-401-2-T 5 assays
EUR 483.6
Description: Our Rac Activation Assays use visible agarose beads to selectively precipitate the active form of Rac1 or Rac2. The precipitated small GTPase is then detected by Western blot using a Rac1- or Rac2-specific antibody included in the kit.

RhoA Activation Assay Kit, Trial Size
STA-403-A-T 5 assays
EUR 483.6
Description: Our Rho Activation Assays use visible agarose beads to selectively precipitate the active form of RhoA, RhoB or RhoC. The precipitated small GTPase is then detected by Western blot using a RhoA-, RhoB- or RhoC-specific antibody included in the kit.

RhoB Activation Assay Kit, Trial Size
STA-403-B-T 5 assays
EUR 483.6
Description: Our Rho Activation Assays use visible agarose beads to selectively precipitate the active form of RhoA, RhoB or RhoC. The precipitated small GTPase is then detected by Western blot using a RhoA-, RhoB- or RhoC-specific antibody included in the kit.

RhoC Activation Assay Kit, Trial Size
STA-403-C-T 5 assays
EUR 483.6
Description: Our Rho Activation Assays use visible agarose beads to selectively precipitate the active form of RhoA, RhoB or RhoC. The precipitated small GTPase is then detected by Western blot using a RhoA-, RhoB- or RhoC-specific antibody included in the kit.

Rap1 Activation Assay Kit, Trial Size
STA-406-1-T 5 assays
EUR 483.6
Description: Our Rap Activation Assays use visible agarose beads to selectively precipitate the active form of Rap1 or Rap2. The precipitated small GTPase is then detected by Western blot using a Rap1- or Rap2-specific antibody included in the kit.

Rap2 Activation Assay Kit, Trial Size
STA-406-2-T 5 assays
EUR 483.6
Description: Our Rap Activation Assays use visible agarose beads to selectively precipitate the active form of Rap1 or Rap2. The precipitated small GTPase is then detected by Western blot using a Rap1- or Rap2-specific antibody included in the kit.

Arf1 Activation Assay Kit, Trial Size
STA-407-1-T 5 assays
EUR 483.6
Description: Our Arf Activation Assays use visible agarose beads to selectively precipitate the active form of Arf1 or Arf 6. The precipitated small GTPase is then detected by Western blot using an Arf1- or Arf6-specific antibody included in the kit.

Arf6 Activation Assay Kit, Trial Size
STA-407-6-T 5 assays
EUR 483.6
Description: Our Arf Activation Assays use visible agarose beads to selectively precipitate the active form of Arf1 or Arf 6. The precipitated small GTPase is then detected by Western blot using an Arf1- or Arf6-specific antibody included in the kit.

Cdc42 Activation Assay Kit, Trial Size
STA-402-T 5 assays
EUR 483.6
Description: Our Cdc42 Activation Assays use visible agarose beads to selectively precipitate the active form of Cdc42 protein. The precipitated small GTPase is then detected by Western blot using a Cdc42-specific antibody included in the kit.

Rac1/Cdc42 Activation Assay Combo Kit
STA-404 2 x 20 assays
EUR 1305.6
Description: Our Rac1/Cdc42 Activation Assays use visible agarose beads to selectively precipitate the active form of the small GTPase. The precipitated small GTPase is then detected by Western blot using a specific antibody included in the kit.

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